Express humanization of antibodies

ABSTRACT

The disclosure provides a method for generation of humanized full length antibodies in mammalian cells. A library of humanized variants is provided with high, validated human framework diversity without requiring back-mutations to retain original affinity. Synthetic CDR encoding fragment libraries derived from a template antibody are ligated to human framework region encoding fragments from a human framework pool limited only to germline sequences from a functionally expressed antibodies. The vector comprises a nucleic acid sequence encoding HC framework region 4. No CDR grafting or phage display is required.

RELATED APPLICATION DATA

This application is a continuation of U.S. patent application Ser. No.16/749,643, filed on Jan. 22, 2020, which, in turn is a continuation ofU.S. patent application Ser. No. 15/593,721, filed on May 12, 2017, nowU.S. Pat. No. 10,562,981, issued on Feb. 18, 2020, which, in turn, is acontinuation of U.S. patent application Ser. No. 13/977,166, filed Jun.28, 2013, now U.S. Pat. No. 9,863,054 issued on Jun. 20, 2017, whichclaims priority to International Application No. PCT/US11/67589, filedDec. 28, 2011, which, in turn, claims the benefit of U.S. ProvisionalApplication No. 61/428,917, filed Dec. 31, 2010, the entire disclosuresof which are hereby incorporated by reference as if set forth fullyherein.

BACKGROUND OF THE INVENTION Field of the Invention

The disclosure provides a method for generation of humanized full lengthantibodies in mammalian cells. A library of humanized variants isprovided with high, validated framework diversity without requiringback-mutations to retain original affinity. Synthetic CDR encodingfragment libraries derived from a template antibody are ligated to humanframework region encoding fragments from a human framework pool limitedonly to germline sequences from a functionally expressed antibodies. Thevector comprises a nucleic acid sequence encoding HC framework region 4.No CDR grafting or phage display is required.

Description of the Related Art

Monoclonal antibodies (MAbs) are monospecific for a particular antigenand are made by identical immune cells that are clones of a uniqueparent cell. Monoclonal antibodies traditionally are made by fusingmyeloma cells with spleen cells from a mouse that has been immunizedwith the desired antigen. Fused hybrid cells, or hybridomas, can begrown indefinitely in cell culture media, or can be injected in micewhere they produce tumors containing an antibody rich fluid calledascites fluid. Antibodies can then be purified from the cell culturemedium or ascites.

Monoclonal antibody therapy is the use of MAbs to specifically bind toan antigen, for example, a cell surface antigen on a target cell. Thismay stimulate the patient's immune system to attack those cells. MAbshave been developed to treat various diseases such as rheumatoidarthritis, multiple sclerosis and different types of cancers. Initially,murine antibodies were obtained with hybridoma technology; however thedissimilarity between murine and human immune systems resulted inseveral clinical failure of these antibodies. Nevertheless, a smallnumber of murine MAbs are FDA approved to treat various conditions.Muromonab-CD3 (Orthoclone OKT3) is a murine MAb that targets the T cellCD3 receptor and was approved in 1986 for transplant rejection.Tositumoman (Bexxar) is a murine Mab that targets CD20 and was approvedin 2003 for the treatment of Non-Hodgkin lymphoma.

Therapeutic deficiencies of mouse monoclonal antibodies as humantherapeutics are well known and include short in vivo half life, weakeffector functions mediated by the mouse heavy chain constant region;patient sensitization to the antibody, and generation of a humananti-mouse antibody (HAMA) response; and neutralization of the mouseantibody by HAMA leading to a loss of therapeutic efficiency. See, forexample, Williams et al. 2010, Humanising antibodies by CDR grafting.Antibody Engineering, Edit by R. Kontermann and S. Dubel, Springer LabManual, 319-339. One major obstacle in early development of therapeuticantibodies was the human anti-murine antibody (HAMA) response whichoccurred in up to about 50% of patients upon administration of murinehybridoma-derived antibodies and compromised the safety, efficacy andhalf-life of the antibody therapeutics.

One way to alleviate certain deficiencies of mouse monoclonal antibodiesis antibody humanization. Various techniques of antibody humanizationare known. One method of antibody humanization is chimerization. Inmouse/human chimeric antibodies, the immunogenic murine constant domainsare replaced by the human counterpart. Intact murine variable domainsare preserved to maintain the intrinsic antigen-binding affinity; i.e.,the entire Fv regions were retained from the murine antibody (about 66%human). Antibody chimerization was found to alleviate the short in vivohalf-life compared to the murine MAb, and impart human Fc effectorfunction on the antibody. Although chimerization of some murineantibodies resulted in reduced HAMA response, others remainedimmunogenic. A few chimeric antibodies are FDA approved to treat variousconditions. Abciximab (ReoPro), is a chimeric antibody which targetsinhibition of glycoprotein IIb/IIIa and was FDA approved in 1994 for thetreatment of cardiovascular disease. Infliximab (Remicade) is a chimericantibody that results in inhibition of TNF-alpha signaling; was firstapproved in 1998 and is now used for the treatment of several autoimmunedisorders.

A second technique of antibody humanization termed CDR grafting involvesthe transplantation of the entire murine CDRs onto a human frameworkregion wherein the reshaped humanized antibody only retained essentialbinding elements from the murine antibody (5-10% of the total sequence).For example, see Lo, Antibody humanization by CDR grafting. AntibodyEngineering, Methods and protocols. Edit by Benny K. C. Lo, Methods inMolecular Biology, 2004, 248, 135-159. CDR grafting is described in U.S.Pat. Nos. 5,225,539 and 5,585,089, each of which is incorporated hereinby reference. Humanized antibodies from CDR grafting resulted inincreased in vivo tolerance and efficacy of therapeutic antibodies.According to Lo 2004, the key to successful CDR grafting lies in thepreservation of the murine CDR conformations in the reshaped antibodyfor antigen binding. The antibody Fv region comprises variable domainsfrom the light chain (V_(L)) and the heavy chain (V_(H)) and confersantibodies with antigen-binding specificity and affinity. The variabledomains adopt the immunoglobulin fold in which two antiparallelbeta-sheet framework scaffolds support three hypervariable CDRs.Unfortunately, CDR grafting often leads to suboptimal orientations ofthe murine CDR loops responsible for antigen binding. Therefore,critical murine framework residues needed to be reintroduced asback-mutations to restore the optimum CDR conformations for antigenbinding. According to Williams et al. 2010, ibid., at least 118antibodies have been humanized. Of the 24 approved antibodies on themarket, 13 are humanized, four are murine, five are chimeric and two arehuman. The marketed, humanized antibodies were all generated by CDRgrafting.

Another technique for developing a minimally immunogenic humanizedantibody is known as SDR grafting. Some humanized antibodies were foundto elicit an anti-idiotype (anti-Id) response against the potentiallyimmunogenic murine CDRs. Further, it was found that not all of the CDRsare equally important, or even essential, for antigen binding. It wasalso found that only about 20-33% of CDR residues are involved inantigen contact. The CDR residues that are most important inantigen-antibody interaction are called specificity-determining residues(SDRs). SDRs are found at positions of high variability and may bedetermined by determination of the three-dimensional structure of theantigen-antibody complex or by genetic manipulation of theantibody-combining site. See for example, Kashmiri et al., 2004,Developing a minimally immunogenic humanized antibody by SDR grafting.Antibody Engineering, Methods and protocols. Edit by Benny K. C. Lo,Methods in Molecular Biology, 248, 361-376. Therefore, there is room forprotein evolution within the CDR regions while maintaining affinity forthe target antigen.

Competing technologies exist to reduce the immunogenicity of antibodies.Transgenic mice (e.g. Xenomouse and UltiMAb-Mouse) containing largeparts of the the human immunoglobulin locus, can be a source of “fullyhuman” antibodies. Human antibodies derived from a bacteriophage libraryof human variable regions also need no humanization, but frequently needfurther mutation to achieve high binding potency. A relatively smallnumber of marketed antibodies have been derived using these otherplatform technologies. See Williams et al. 2010, ibid.

According to Lo 2004, the goal of antibody humanization is to engineer amonoclonal antibody (MAb) raised in a non-human species into one that isless immunogenic when administered to humans. However, it would beadvantageous to develop a technique of antibody humanization that isaccompanied by various protein evolutionary techniques to produce ahumanized antibody with other optimized characteristics such as enhancedaffinity for the target antigen compared to the mouse and/or increasedexpression.

SUMMARY OF THE INVENTION

The disclosure provides methods of express, rapid humanization ofantibodies. One or more libraries of full length antibodies aregenerated and simultaneously screened for binding and expressionoptimization. The antibody libraries are specifically designed to besmall in member number, but highly diverse. Only validated humanframeworks are employed in the antibody libraries, which have beenfunctionally expressed from germline sequences. A subset of germlinesequences are specifically selected for maximum diversity in the finallibrary. In one aspect, a single sequence for LC and HC Framework 4 isemployed for the entire library, and DNA encoding this frameworksequence is built into the vector. Ligation is used to recombineframeworks and CDRs, thus avoiding overlap PCR which requires multipleprimer sets. In one aspect, the library is expressed and screened in themanufacturing host. In another aspect, an antibody equal to or superiorto the donor antibody, for example a mouse MAb, in terms of antigenaffinity is identified in three to four months.

In one embodiment, the disclosure provides a method of producing ahumanized antibody comprising the step of synthesizing immunoglobulinheavy chain (HC) double stranded DNA fragment libraries comprisingcomplementarity determining region (CDR) fragment encoding libraries andframework (FW) fragment encoding libraries, wherein at least one CDRfragment library is derived from the template antibody and each FWfragment library is from a human framework pool obtained fromfunctionally expressed human antibodies.

In one aspect, the disclosure provides a method of producing a humanizedantibody comprising the step of synthesizing immunoglobulin light chain(LC) double stranded DNA fragment libraries comprising CDR fragmentencoding libraries and FW fragment encoding libraries, wherein at leastone CDR fragment library is derived from the template antibody and eachFW fragment library is from a human framework pool obtained fromfunctionally expressed human antibodies.

In another aspect, the disclosure provides a method of producing ahumanized antibody comprising the step of producing a humanized antibodyfurther comprises assembling from the HC fragment libraries by stepwiseliquid phase ligation of heavy chain FW encoding fragments and CDRencoding fragments in the order of: FW1-CDR1-FW2-CDR2-FW3-CDR3 toproduce a humanized HC variable domain encoding library.

In a further aspect, the disclosure provides a method of producing ahumanized antibody comprising the step of producing a humanized antibodyfurther comprises assembling from the LC fragment libraries by stepwiseliquid phase ligation of light chain FW encoding fragments and CDRencoding fragments in the order of: FW1-CDR1-FW2-CDR2-FW3-CDR3 toproduce a humanized LC variable domain encoding library.

In another aspect, the disclosure provides a method of producing ahumanized antibody comprising the steps of cloning the assembledhumanized heavy chain variable domain library and the assembled lightchain variable domain library into an expression vector to create ahumanization library; transfecting the humanization library into cells;and expressing full length humanized antibodies in the cells to create ahumanized antibody library.

In further aspects, the disclosure provides a method of producing ahumanized antibody comprising the steps of screening the humanizedantibody library to determine the expression level of the humanizedantibodies; and screening the humanized antibody library to determinethe affinity of the humanized antibodies for the antigen compared to theaffinity of the template antibody to the antigen. In one aspect, thehumanized antibody exhibits equal or greater affinity for an antigencompared to a template antibody.

In one aspect, the disclosure provides a method of producing a humanizedantibody comprising the step of cloning the assembled humanized heavychain variable domain library and the assembled light chain variabledomain library into an expression vector comprising a nucleic acidsequence encoding HC framework region 4. In a specific aspect, thenucleotide sequence encoding framework 4 is derived from a human heavychain variable domain derived from a functionally expressed humanantibody.

In another aspect, the vector comprises a nucleic acid sequence encodingLC framework region 4. In a specific aspect, the nucleotide sequenceencoding framework 4 is derived from a human light chain variable domainderived from a functionally expressed human antibody.

In various aspects, the disclosure provides a method of producing ahumanized antibody library wherein the member number of the humanizedantibody library is 10,000,000 members or fewer; 1,000,000 members orfewer; or 100,000 members or fewer.

In one aspect, the disclosure provides a method of producing a humanizedantibody comprising the step of cloning the assembled humanized HCvariable domain library into an expression vector to create a vector-HCvariable domain DNA library, and ligating the assembled light chainvariable domain library into the vector-HC library to create thehumanization library. In another aspect, the expression step comprisesexpressing both the humanized heavy chain variable domain and thehumanized light chain variable domain from a single promoter.

In another aspect, the disclosure provides a method of producing ahumanized antibody comprising a step of screening the humanized antibodylibrary for a humanized antibody having one or more additional improvedcharacteristics when compared to the template antibody; the one or morecharacteristics selected from the group consisting of: equilibriumdissociation constant (K_(D)); stability; melting temperature (T_(m));pI; solubility; expression level; reduced immunogenicity and improvedeffector function. In certain aspects, the improvement is between about1% and 500%, relative to the template antibody or is between about 2fold and 1000 fold relative to the template antibody.

In another aspect, the disclosure provides a method of producing ahumanized antibody further comprising the step of cloning the assembledhumanized heavy chain variable domain library and the assembled lightchain variable domain library into an expression vector to create ahumanization library; and transfecting the humanization library intocells; wherein the cells are selected from a eukaryotic cell productionhost cell line selected from a member of the group consisting of 3T3mouse fibroblast cells; BHK21 Syrian hamster fibroblast cells; MDCK, dogepithelial cells; Hela human epithelial cells; PtK1 rat kangarooepithelial cells; SP2/0 mouse plasma cells; and NS0 mouse plasma cells;HEK 293 human embryonic kidney cells; COS monkey kidney cells; CHO,CHO-S Chinese hamster ovary cells; R1 mouse embryonic cells; E14.1 mouseembryonic cells; H1 human embryonic cells; H9 human embryonic cells; PERC.6, human embryonic cells; S. cerevisiae yeast cells; and picchia yeastcells. In specific aspects, the eukaryotic cell production host cellline is selected from CHO-S; HEK293; CHOK1SV or NS0. In another aspect,the cell is a eukaryotic cell production host with antibody cell surfacedisplay and, optionally, one or more of the screening steps areperformed in the eukaryotic cell production host.

In one embodiment, the disclosure provides a method of producing ahumanized antibody comprising screening a humanized library utilizing anassay selected from quantitative ELISA; affinity ELISA; ELISPOT; flowcytometry, immunocytology, Biacore® surface plasmon resonance analysis,Sapidyne KinExA™ kinetic exclusion assay; SDS-PAGE; Western blot, orHPLC.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic of the method of rapid antibody humanization ofa template antibody.

FIG. 2 shows data from the primary screen of humanized variants oftemplate antibody BA001. The primary screen comprised high throughputELISA of variants compared to the donor antibody, shown with anasterisk. ELISAs were used to determine antigen binding andquantitation.

FIG. 3 shows the top 8 confirmed humanized antibody variant hits interms of both expression and function compared to the template antibodyBA001. DNA and protein sequences for BA001 and humanized variants shownare described in U.S. Publication No. US 2010/0138945, which isincorporated herein by reference.

FIG. 4 shows binding affinity BiaCore surface plasmon resonance data forhumanized anti-IL6 antibodies compared to a template antibody. Data forthe template, CNT0328, a chimeric, human-murine antibody, is from US2006/0257407, which is incorporated herein by reference. BA001 is also atemplate antibody that has the same sequence as the template CNT0328,but was manufactured in a different expression system. Humanized variantantibodies h1-h8 were obtained with no additional affinity maturation.

FIG. 5 shows results of an ELISA wherein humanized antibody variantsblock antigen binding of template antibody BA001.

DEFINITION OF TERMS

In order to facilitate understanding of the examples provided herein,certain frequently occurring methods and/or terms will be described.

The term “affinity maturation” refers to the increase in averageaffinity of an immune response for an antigen. In nature, it can occurafter repeated exposure to an antigen. A particularly preferred type ofsubstitutional variant involves substituting one or more hypervariableregion residues of a parent antibody (e.g. human antibody). Generally,the resulting variant(s) selected for further development will haveimproved biological properties relative to the parent antibody fromwhich they are generated. A convenient way for generating suchsubstitutional variants involves affinity maturation using techniquesdescribed herein or other techniques known to one of skill in the art,for example, phage display (Schier R., J. Mol. Biol., 263:551-67, 1996).The variants are then screened for their biological activity (e.g.binding affinity) as described herein, e.g. Biacore analysis. In orderto identify hypervariable region residues which would be good candidatesfor modification, alanine scanning mutagenesis can be performed toidentify hypervariable region residues contributing significantly toantigen binding. Antibodies with superior properties in one or morerelevant assays can undergo further development.

The term “agent” is used herein to denote an antibody or antibodylibrary. Agents are evaluated for potential activity as, for example,anti-neoplastics, anti-inflammatories or apoptosis modulators byinclusion in screening assays described hereinbelow. Agents areevaluated for potential activity as specific protein interactioninhibitors (i.e., an agent which selectively inhibits a bindinginteraction between two predetermined polypeptides but which does notsubstantially interfere with cell viability) by inclusion in screeningassays described hereinbelow.

The term “amino acid” as used herein refers to any organic compound thatcontains an amino group (—NH₂) and a carboxyl group (—COOH); preferablyeither as free groups or alternatively after condensation as part ofpeptide bonds. The “twenty naturally encoded polypeptide-formingalpha-amino acids” are understood in the art and refer to: alanine (alaor A), arginine (arg or R), asparagine (asn or N), aspartic acid (asp orD), cysteine (cys or C), gluatamic acid (glu or E), glutamine (gln orQ), glycine (gly or G), histidine (his or H), isoleucine (ile or I),leucine (leu or L), lysine (lys or K), methionine (met or M),phenylalanine (phe or F), proline (pro or P), serine (ser or S),threonine (thr or T), tryptophan (trp or W), tyrosine (tyr or Y), andvaline (val or V).

The term “amplification” means that the number of copies of apolynucleotide is increased.

The term “antibody”, as used herein, refers to intact immunoglobulinmolecules, as well as fragments of immunoglobulin molecules, such asFab, Fab′, (Fab′)2, Fv, and SCA fragments, that are capable of bindingto an epitope of an antigen.

An Fab fragment consists of a monovalent antigen-binding fragment of anantibody molecule, and can be produced by digestion of a whole antibodymolecule with the enzyme papain, to yield a fragment consisting of anintact light chain and a portion of a heavy chain.

An Fab′ fragment of an antibody molecule can be obtained by treating awhole antibody molecule with pepsin, followed by reduction, to yield amolecule consisting of an intact light chain and a portion of a heavychain. Two Fab′ fragments are obtained per antibody molecule treated inthis manner.

An (Fab′)2 fragment of an antibody can be obtained by treating a wholeantibody molecule with the enzyme pepsin, without subsequent reduction.A (Fab′)2 fragment is a dimer of two Fab′ fragments, held together bytwo disulfide bonds.

An Fv fragment is defined as a genetically engineered fragmentcontaining the variable region of a light chain and the variable regionof a heavy chain expressed as two chains.

A single chain antibody (“SCA”) is a genetically engineered single chainmolecule containing the variable region of a light chain and thevariable region of a heavy chain, linked by a suitable, flexiblepolypeptide liner.

The term “biosimilar”, also termed “follow-on biologic”, refers toofficially approved new versions of innovator biopharmaceuticalproducts, following patent or exclusivity expiry.

The term “cell production host”, or “manufacturing host”, refers to acell line used for the production or manufacturing of proteins.Eukaryotic cells such as mammalian cells, including, but not limited tohuman, mouse, hamster, rat, monkey cell lines as well as yeast, insectand plant cell lines. Prokaryotic cells can alternatively be utilized.In one aspect, a mammalian cell production host is selected from amember of the group consisting of 3T3 mouse fibroblast cells; BHK21Syrian hamster fibroblast cells; MDCK, dog epithelial cells; Hela humanepithelial cells; PtK1 rat kangaroo epithelial cells; SP2/0 mouse plasmacells; and NS0 mouse mouse plasma cells; HEK 293 human embryonic kidneycells; COS monkey kidney cells; CHO, CHO-S Chinese hamster ovary cells;R1 mouse embryonic cells; E14.1 mouse embryonic cells; H1 humanembryonic cells; H9 human embryonic cells; PER C.6, human embryoniccells. In another aspect, the cell production host is a GS-NS0 orGS-CHOK1 cell line. In another aspect, the cell production host isselected from S. cerevisiae yeast cells; and picchia yeast cells. Inanother aspect, the cell production host is a bacterial cell line.

A molecule that has a “chimeric property” is a molecule that is: 1) inpart homologous and in part heterologous to a first reference molecule;while 2) at the same time being in part homologous and in partheterologous to a second reference molecule; without 3) precluding thepossibility of being at the same time in part homologous and in partheterologous to still one or more additional reference molecules. In anon-limiting embodiment, a chimeric molecule may be prepared byassemblying a reassortment of partial molecular sequences. In anon-limiting aspect, a chimeric polynucleotide molecule may be preparedby synthesizing the chimeric polynucleotide using plurality of moleculartemplates, such that the resultant chimeric polynucleotide hasproperties of a plurality of templates.

The term “cognate” as used herein refers to a gene sequence that isevolutionarily and functionally related between species. For example,but not limitation, in the human genome the human CD4 gene is thecognate gene to the mouse 3d4 gene, since the sequences and structuresof these two genes indicate that they are highly homologous and bothgenes encode a protein which functions in signaling T cell activationthrough MHC class II-restricted antigen recognition.

The term “commercial scale” means production of a protein or antibody ata scale appropriate for resale.

A “comparison window,” as used herein, refers to a conceptual segment ofat least 20 contiguous nucleotide positions wherein a polynucleotidesequence may be compared to a reference sequence of at least 20contiguous nucleotides and wherein the portion of the polynucleotidesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. Optimal alignment of sequences for aligning acomparison window may be conducted by the local homology algorithm ofSmith and Waterman (1981) Adv. Appl. Math. 2: 482 by the homologyalignment algorithm of Needlemen and Wuncsch J. Mol. Biol. 48: 443(1970), by the search of similarity method of Pearson and Lipman Proc.Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package Release 7.0, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by inspection, and the bestalignment (i.e., resulting in the highest percentage of homology overthe comparison window) generated by the various methods is selected.

As used herein, the term “complementarity-determining region” and “CDR”refer to the art-recognized term as exemplified by the Kabat andChothia. CDR definitions are also generally known as supervariableregions or hypervariable loops (Chothia and Leks, 1987; Clothia et al.,1989; Kabat et al., 1987; and Tramontano et al., 1990). Variable regiondomains typically comprise the amino-terminal approximately 105-115amino acids of a naturally-occurring immunoglobulin chain (e.g., aminoacids 1-110), although variable domains somewhat shorter or longer arealso suitable for forming single-chain antibodies. The CDRs are parts ofimmunoglobulins that determine the specificity of said molecules andmake contact with a specific ligand. The CDRs are the most variable partof the molecule and contribute to the diversity of these molecules.There are three CDR regions CDR1, CDR2 and CDR3 in each V domain CDR-Hdepicts a CDR region of a variable heavy chain and CDR-L relates to aCDR region of a variable light chain. H means the variable heavy chainand L means the variable light chain. The CDR regions of an Ig-derivedregion may be determined as described in Kabat (1991). Sequences ofProteins of Immunological Interest, 5th edit., NIH Publication no.91-3242 U.S. Department of Health and Human Services, Chothia (1987) J.Mol. Biol. 196, 901-917 and Chothia (1989) Nature, 342, 877-883.

The term “comprehensive” is used herein to refer to a technique ofevolution wherein every possible change is made at each position of atemplate polynucleotide or template polypeptide and the polynucleotideor polypeptide is tested to confirm the intended change has been made bysequencing or some other technique. Comprehensive mutagenesis refers tomutating the DNA of a region of a gene encoding a protein that changescodon amino acid sequence of the protein and then determining viasequencing or other technologies that all mutations have been made andin the optimal case arrayed where every clone is in an identifiableposition and/or uniquely tagged. Then screening of all of the expressedmutants is performed to ensure that all are expressed comprehensivelyfor an improved phenotype in order to provide guaranteed comprehensivecoverage, i.e. CPE library with Comprehensive Screening comprising theBioAtla CPE process. Non-expressing clones in the screening system willalso be simultaneously measured for expression to ensure that are notincorrectly labeled as negative or neutral mutations once enabled forexpression an alternative system such as in vitro transcription andtranslation. Alternatively, sequencing could be performed on all clonesafter screening, but it should include all negative, neutral andup-mutant clones. Any mutants not identified are then be added in asecond round of screening to yield and a true comprehensive mutagenesisand screening expression/activity system such as CPE. This is enabled inpart by recent successes in high throughput sequencing that did notexist previously.

The term “Comprehensive Positional Evolution” (CPE™) is used to describean antibody evolution technology platform that can be used to combinecomprehensive mutagenesis, shuffling and synthesis technologies toenhance single or multiple antibody properties and bindingcharacteristics. The CPE platform allows for the comprehensive mappingof the in vivo effects of every individual codon change within theprotein for all 63 potential codon changes at each position within theprotein. This comprehensive mutagenesis technology rapidly generatesantibody variants by testing amino acid changes at every position alongan antibody variable domain's sequence.

The term “Combinatorial Protein Synthesis” (CPS™) is used to describecombinatorial protein synthesis technologies that can be used tooptimize the desired characteristics of antibodies by combining theirbest properties into a new, high-performance antibody. CPS™ can be usedfollowing CPE™ and can allow for the subsequent generation and in vivoselection of all permutations of improved individual codons foridentification of the optimal combination or set of codon changes withina protein or antibody. The combination of these technologies cansignificantly expand the pool of antibody variants available to bescreened and it significantly increases the probability of findingantibodies with single or multiple enhanced characteristics such asbinding affinity, specificity, thermo-stability, expression level,effector function, glycosylation, and solubility.

For full length antibody molecules, the immunoglobulin genes can beobtained from genomic DNA or mRNA of hybridoma cell lines. Antibodyheavy and light chains are cloned in a mammalian vector system. Assemblyis documented with double strand sequence analysis. The antibodyconstruct can be expressed in other human or mammalian host cell lines.The construct can then be validated by transient transfection assays andWestern blot analysis of the expressed antibody of interest. Stable celllines with the highest productivity can be isolated and screened usingrapid assay methods.

“Conservative amino acid substitutions” refer to the interchangeabilityof residues having similar side chains. For example, a group of aminoacids having aliphatic side chains is glycine, alanine, valine, leucine,and isoleucine; a group of amino acids having aliphatic-hydroxyl sidechains is serine and threonine; a group of amino acids havingamide-containing side chains is asparagine and glutamine; a group ofamino acids having aromatic side chains is phenylalanine, tyrosine, andtryptophan; a group of amino acids having basic side chains is lysine,arginine, and histidine; and a group of amino acids havingsulfur-containing side chains is cysteine and methionine. Preferredconservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine.

The term “corresponds to” is used herein to mean that a polynucleotidesequence is homologous (i.e., is identical, not strictly evolutionarilyrelated) to all or a portion of a reference polynucleotide sequence, orthat a polypeptide sequence is identical to a reference polypeptidesequence. In contradistinction, the term “complementary to” is usedherein to mean that the complementary sequence is homologous to all or aportion of a reference polynucleotide sequence. For illustration, thenucleotide sequence “TATAC” corresponds to a reference “TATAC” and iscomplementary to a reference sequence “GTATA.”

The term “degrading effective” amount refers to the amount of which isrequired to process at least 50% of the substrate, as compared tosubstrate not contacted with the enzyme. Preferably, at least 80% of thesubstrate is degraded.

As used herein, the term “defined sequence framework” refers to a set ofdefined sequences that are selected on a non-random basis, generally onthe basis of experimental data or structural data; for example, adefined sequence framework may comprise a set of amino acid sequencesthat are predicted to form a (3-sheet structure or may comprise aleucine zipper heptad repeat motif, a zinc-finger domain, among othervariations. A “defined sequence kernal” is a set of sequences whichencompass a limited scope of variability. Whereas (1) a completelyrandom 10-mer sequence of the 20 conventional amino acids can be any of(20)10 sequences, and (2) a pseudorandom 10-mer sequence of the 20conventional amino acids can be any of (20)10 sequences but will exhibita bias for certain residues at certain positions and/or overall, (3) adefined sequence kernal is a subset of sequences if each residueposition was allowed to be any of the allowable 20 conventional aminoacids (and/or allowable unconventional amino/imino acids). A definedsequence kernal generally comprises variant and invariant residuepositions and/or comprises variant residue positions which can comprisea residue selected from a defined subset of amino acid residues), andthe like, either segmentally or over the entire length of the individualselected library member sequence. Defined sequence kernels can refer toeither amino acid sequences or polynucleotide sequences. Of illustrationand not limitation, the sequences (NNK)10 and (NNM)10, wherein Nrepresents A, T, G, or C; K represents G or T; and M represents A or C,are defined sequence kernels.

The term “deimmunization” as used herein relates to production of avariant of the template binding molecule, which is modified compared toan original wild type molecule by rendering said variant non-immunogenicor less immunogenic in humans. Deimmunized molecules according to theinvention relate to antibodies or parts thereof (like frameworks and/orCDRs) of non-human origin. Corresponding examples are antibodies orfragments thereof as described in U.S. Pat. No. 4,361,549. The term“deimmunized” also relates to molecules, which show reduced propensityto generate T cell epitopes. In accordance with this invention, the term“reduced propensity to generate T cell epitopes” relates to the removalof T-cell epitopes leading to specific T-cell activation.

Furthermore, reduced propensity to generate T cell epitopes meanssubstitution of amino acids contributing to the formation of T cellepitopes, i.e. substitution of amino acids, which are essential forformation of a T cell epitope. In other words, reduced propensity togenerate T cell epitopes relates to reduced immunogenicity or reducedcapacity to induce antigen independent T cell proliferation. Inaddition, reduced propensity to generate T cell epitopes relates todeimmunization, which means loss or reduction of potential T cellepitopes of amino acid sequences inducing antigen independent T cellproliferation.

The term “T cell epitope” as used herein relates to short peptidesequences which can be released during the degradation of peptides,polypeptide or proteins within cells and subsequently be presented bymolecules of the major histocompatibility complex (MHC) in order totrigger the activation of T cells; see inter alia WO 02/066514. Forpeptides presented by MHC class II such activation of T cells can theninduce an antibody response by direct stimulation of B cells to producesaid antibodies.

“Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a gel to isolate the desired fragment.

The term “DNA shuffling” is used herein to indicate recombinationbetween substantially homologous but non-identical sequences, in someembodiments DNA shuffling may involve crossover via non-homologousrecombination, such as via cer/lox and/or flp/frt systems and the like.Shuffling may be random or non-random.

As used in this invention, the term “epitope” refers to an antigenicdeterminant on an antigen, such as a phytase polypeptide, to which theparatope of an antibody, such as a phytase-specific antibody, binds.Antigenic determinants usually consist of chemically active surfacegroupings of molecules, such as amino acids or sugar side chains, andcan have specific three-dimensional structural characteristics, as wellas specific charge characteristics. As used herein “epitope” refers tothat portion of an antigen or other macromolecule capable of forming abinding interaction that interacts with the variable region binding bodyof an antibody. Typically, such binding interaction is manifested as anintermolecular contact with one or more amino acid residues of a CDR.

The term “evolution” refers to a change in at least one property,characteristic or activity of a genetically or synthetically modifiedantibody when compared to a template antibody.

The terms “fragment”, “derivative” and “analog” when referring to areference polypeptide comprise a polypeptide which retains at least onebiological function or activity that is at least essentially same asthat of the reference polypeptide. Furthermore, the terms “fragment”,“derivative” or “analog” are exemplified by a “pro-form” molecule, suchas a low activity proprotein that can be modified by cleavage to producea mature enzyme with significantly higher activity.

The term “fragment” when applied to a nucleic acid sequence refers to amolecule that encodes for a portion, or a sub-portion, of an antibodymolecule. For example, an HC CDR1 DNA fragment, may encode the entireheavy chain CDR1, or a truncated portion thereof.

In one aspect, certain methods provided herein provide for producingfrom a template polypeptide a set of progeny polypeptides in which a“full range of single amino acid substitutions” is represented at eachamino acid position. As used herein, “full range of single amino acidsubstitutions” is in reference to the naturally encoded 20 naturallyencoded polypeptide-forming alpha-amino acids, as described herein.

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

“Genetic instability”, as used herein, refers to the natural tendency ofhighly repetitive sequences to be lost through a process of reductiveevents generally involving sequence simplification through the loss ofrepeated sequences. Deletions tend to involve the loss of one copy of arepeat and everything between the repeats.

The term “heterologous” means that one single-stranded nucleic acidsequence is unable to hybridize to another single-stranded nucleic acidsequence or its complement. Thus, areas of heterology means that areasof polynucleotides or polynucleotides have areas or regions within theirsequence which are unable to hybridize to another nucleic acid orpolynucleotide. Such regions or areas are for example areas ofmutations.

The term “homologous” or “homeologous” means that one single-strandednucleic acid nucleic acid sequence may hybridize to a complementarysingle-stranded nucleic acid sequence. The degree of hybridization maydepend on a number of factors including the amount of identity betweenthe sequences and the hybridization conditions such as temperature andsalt concentrations as discussed later. Preferably the region ofidentity is greater than about 5 bp, more preferably the region ofidentity is greater than 10 bp.

The term “humanized” is used to describe antibodies whereincomplementarity determining regions (CDRs) from a mammalian animal,e.g., a mouse, are combined with a human framework region. Oftenpolynucleotides encoding the isolated CDRs will be grafted intopolynucleotides encoding a suitable variable region framework (andoptionally constant regions) to form polynucleotides encoding completeantibodies (e.g., humanized or fully-human), antibody fragments, and thelike. In another aspect, besides mouse antibodies, other species can behumanized, such as, for example, other rodent, camel, rabbit, cat, dog,pig, horse, cow, fish, llama and shark. In a broad aspect, any speciesthat produces antibodies can be utilized in the production of humanizedantibodies. Additionally, the antibodies of the invention may bechimeric, human-like, humanized or fully human, in order to reduce theirpotential antigenicity, without reducing their affinity for theirtarget. Chimeric, human-like and humanized antibodies have generallybeen described in the art. By incorporating as little foreign sequenceas possible in the hybrid antibody, the antigenicity is reduced.Preparation of these hybrid antibodies may be carried out by methodswell known in the art.

An immunoglobulin light or heavy chain variable region consists of a“framework” region interrupted by three hypervariable regions, alsocalled CDR's. The extent of the framework region and CDR's have beenprecisely defined (see, “Sequences of Proteins of ImmunologicalInterest,” Kabat et al., 1987). The sequences of the framework regionsof different light or heavy chains are relatively conserved within aspecies. As used herein, a “human framework region” is a frameworkregion that is substantially identical (about 85 or more, usually 90-95or more) to the framework region of a naturally occurring humanimmunoglobulin. The framework region of an antibody, that is thecombined framework regions of the constituent light and heavy chains,serves to position and align the CDR's. The CDR's are primarilyresponsible for binding to an epitope of an antigen. In accordance withthis invention, a framework region relates to a region in the V domain(VH or VL domain) of immunoglobulins that provides a protein scaffoldfor the hypervariable complementarity determining regions (CDRs) thatmake contact with the antigen. In each V domain, there are fourframework regions designated FR1, FR2, FR3 and FR4. Framework 1encompasses the region from the N-terminus of the V domain until thebeginning of CDR1, framework 2 relates to the region between CDR1 andCDR2, framework 3 encompasses the region between CDR2 and CDR3 andframework 4 means the region from the end of CDR3 until the C-terminusof the V domain; see, inter alia, Janeway, Immunobiology, GarlandPublishing, 2001, 5th ed. Thus, the framework regions encompass all theregions outside the CDR regions in VH or VL domains. In one aspect ofthe disclosure, a single sequence is employed for framework 4 which isheld constant through each member of the antibody library. In oneaspect, the single sequence encoding framework region 4 is the mostcommon sequence found in a human framework pool limited only to germlinesequences from a functionally expressed antibodies.

The person skilled in the art is readily in a position to deduce from agiven sequence the framework regions and, the CDRs; see Kabat (1991)Sequences of Proteins of Immunological Interest, 5th edit., NIHPublication no. 91-3242 U.S. Department of Health and Human Services,Chothia (1987) J. Mol. Biol. 196, 901-917 and Chothia (1989) Nature,342, 877-883.

The benefits of this invention extend to “industrial applications” (orindustrial processes), which term is used to include applications incommercial industry proper (or simply industry) as well asnon-commercial industrial applications (e.g. biomedical research at anon-profit institution). Relevant applications include those in areas ofdiagnosis, medicine, agriculture, manufacturing, and academia.

The term “identical” or “identity” means that two nucleic acid sequenceshave the same sequence or a complementary sequence. Thus, “areas ofidentity” means that regions or areas of a polynucleotide or the overallpolynucleotide are identical or complementary to areas of anotherpolynucleotide or the polynucleotide.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide or proteinpresent in a living animal is not isolated, but the same polynucleotideor protein, separated from some or all of the coexisting materials inthe natural system, is isolated. Such polynucleotides could be part of avector and/or such polynucleotides or proteins could be part of acomposition, and still be isolated in that such vector or composition isnot part of its natural environment.

By “isolated nucleic acid” is meant a nucleic acid, e.g., a DNA or RNAmolecule, that is not immediately contiguous with the 5′ and 3′ flankingsequences with which it normally is immediately contiguous when presentin the naturally occurring genome of the organism from which it isderived. The term thus describes, for example, a nucleic acid that isincorporated into a vector, such as a plasmid or viral vector; a nucleicacid that is incorporated into the genome of a heterologous cell (or thegenome of a homologous cell, but at a site different from that at whichit naturally occurs); and a nucleic acid that exists as a separatemolecule, e.g., a DNA fragment produced by PCR amplification orrestriction enzyme digestion, or an RNA molecule produced by in vitrotranscription. The term also describes a recombinant nucleic acid thatforms part of a hybrid gene encoding additional polypeptide sequencesthat can be used, for example, in the production of a fusion protein.

As used herein “ligand” refers to a molecule, such as a random peptideor variable segment sequence, that is recognized by a particularreceptor. As one of skill in the art will recognize, a molecule (ormacromolecular complex) can be both a receptor and a ligand. In general,the binding partner having a smaller molecular weight is referred to asthe ligand and the binding partner having a greater molecular weight isreferred to as a receptor.

“Ligation” refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Maniatis et al., 1982, p.146). Unless otherwise provided, ligation may be accomplished usingknown buffers and conditions with 10 units of T4 DNA ligase (“ligase”)per 0.5 μg of approximately equimolar amounts of the DNA fragments to beligated.

As used herein, “linker” or “spacer” refers to a molecule or group ofmolecules that connects two molecules, such as a DNA binding protein anda random peptide, and serves to place the two molecules in a preferredconfiguration, e.g., so that the random peptide can bind to a receptorwith minimal steric hindrance from the DNA binding protein.

The term “mammalian cell surface display” refers to a technique wherebya protein or antibody, or a portion of an antibody, is expressed anddisplayed on a mammalian host cell surface for screening purposes; forexample, by screening for specific antigen binding by a combination ofmagnetic beads and fluorescence-activated cell sorting. In one aspect,mammalian expression vectors are used for simultaneous expression ofimmunoglobulins as both a secreted and cell surface bound form as inDuBridge et al., US 2009/0136950, which is incorporated herein byreference. In another aspect, the techniques of Gao et al. are employedfor a viral vector encoding for a library of antibodies or antibodyfragments are displayed on the cell membranes when expressed in a cellas in Gao et al., US 2007/0111260, incorporated herein by reference.Whole IgG surface display on mammalian cells is known. For example, aAkamatsuu et al. developed a mammalian cell surface display vector,suitable for directly isolating IgG molecules based on theirantigen-binding affinity and biological activity. Using an Epstein-Barrvirus-derived episomal vector, antibody libraries were displayed aswhole IgG molecules on the cell surface and screened for specificantigen binding by a combination of magnetic beads andfluorescence-activated cell sorting. Plasmids encoding antibodies withdesired binding characteristics were recovered from sorted cells andconverted to the form for production of soluble IgG. Akamatsuu et al. J.Immunol. Methods 2007 327(1-2):40-52; incorporated herein by reference.Ho et al. used human embryonic kidney 293T cells that are widely usedfor transient protein expression for cell surface display ofsingle-chain Fv antibodies for affinity maturation. Cells expressing arare mutant antibody with higher affinity were enriched 240-fold by asingle-pass cell sorting from a large excess of cells expressing WTantibody with a slightly lower affinity. Furthermore, a highly enrichedmutant was obtained with increased binding affinity for CD22 after asingle selection of a combinatory library randomizing an intrinsicantibody hotspot. Ho et al. Isolation of anti-CD22 Fv with high affinityby Fv display on human cells, Proc Natl Acad Sci USA 2006 Jun. 20;103(25): 9637-9642; incorporated herein by reference.

Beerli et al. used B cells specific for an antigen of interest whichwere directly isolated from peripheral blood mononuclear cells (PBMC) ofhuman donors. Recombinant, antigen-specific single-chain Fv (scFv)libraries are generated from this pool of B cells and screened bymammalian cell surface display by using a Sindbis virus expressionsystem. This method allows isolating antigen-specific antibodies by asingle round of FACS. The variable regions (VRs) of the heavy chains(HCs) and light chains (LCs) were isolated from positive clones andrecombinant fully human antibodies produced as whole IgG or Fabfragments. In this manner, several hypermutated high-affinity antibodiesbinding the Qβ virus like particle (VLP), a model viral antigen, as wellas antibodies specific for nicotine were isolated. All antibodies showedhigh expression levels in cell culture. The human nicotine-specific mAbswere validated preclinically in a mouse model. Beerli et al., Isolationof human monoclonal antibodies by mammalian cell display, Proc Natl AcadSci USA. 2008 Sep. 23; 105(38): 14336-14341; incorporated herein byreference.

Yeast cell surface display is also known, for example, see Kondo andUeda 2004, Yeast cell-surface display-applications of molecular display,Appl. Microbiol. Biotechnol., 64(1): 28-40, which describes for example,a cell-surface engineering system using the yeast Saccharomycescerevisiae. Several representative display systems for the expression inyeast S. cerevisiae are described in Lee et al, 2003, Microbialcell-surface display, TRENDS in Bitechnol. 21(1): 45-52. Also Boder andWittrup 1997, Yeast surface display for screening combinatorialpolypeptide libraries, Nature Biotechnol., 15(6): 553.

The term “manufacturing” refers to production of a protein at asufficient quantity to permit at least Phase I clinical testing of atherapeutic protein, or sufficient quantity for regulatory approval of adiagnostic protein.

The term “missense mutation” refers to a point mutation where a singlenucleotide is changed, resulting in a codon that codes for a differentamino acid. Mutations that change an amino acid to a stop codon arecalled nonsense mutations.

As used herein, a “molecular property to be evolved” includes referenceto molecules comprised of a polynucleotide sequence, molecules comprisedof a polypeptide sequence, and molecules comprised in part of apolynucleotide sequence and in part of a polypeptide sequence.Particularly relevant—but by no means limiting—examples of molecularproperties to be evolved include enzymatic activities at specifiedconditions, such as related to temperature; salinity; pressure; pH; andconcentration of glycerol, DMSO, detergent, and/or any other molecularspecies with which contact is made in a reaction environment. Additionalparticularly relevant—but by no means limiting—examples of molecularproperties to be evolved include stabilities—e.g., the amount of aresidual molecular property that is present after a specified exposuretime to a specified environment, such as may be encountered duringstorage.

The term “Multidimensional Epitope Mapping” (MEM) refers to theidentification of the epitope and the resolution of the amino acids thatare important for antibody binding. Information about the binding sites(epitopes) of proteins recognized by antibodies is important for theiruse as biological or diagnostic tools as well as for understanding theirmechanisms of action. However, antigens are highly diverse, in theirprimary sequence as well as in three dimensional structures. Epitopesgenerally fall into 3 categories: 1) linear epitopes, i.e. the antibodybinds to residues on a linear part of the polypeptide chain, 2)conformational epitopes, where the binding site is formed by astructural element (e.g. α-helix, loop), 3) discontinuous epitopes wheretwo or more separate stretches of the polypeptide chain which arebrought together in the three dimensional structure of the antigen formthe binding surface.

The term “mutating” refers to creating a mutation in a nucleic acidsequence; in the event where the mutation occurs within the codingregion of a protein, it will lead to a codon change which may or may notlead to an amino acid change.

The term “mutations” means changes in the sequence of a wild-typenucleic acid sequence or changes in the sequence of a peptide orpolypeptides. Such mutations may be point mutations such as transitionsor transversions. The mutations may be deletions, insertions orduplications.

As used herein, the degenerate “N,N,G/T” nucleotide sequence represents32 possible triplets, where “N” can be A, C, G or T.

As used herein, the degenerate “N,N,N” nucleotide sequence represents 64possible triplets, where “N” can be A, C, G or T.

The term “naturally-occurring” as used herein as applied to the objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally occurring. Generally, the term naturally occurring refers toan object as present in a non-pathological (un-diseased) individual,such as would be typical for the species.

As used herein, a “nucleic acid molecule” is comprised of at least onebase or one base pair, depending on whether it is single-stranded ordouble-stranded, respectively. Furthermore, a nucleic acid molecule maybelong exclusively or chimerically to any group of nucleotide-containingmolecules, as exemplified by, but not limited to, the following groupsof nucleic acid molecules: RNA, DNA, genomic nucleic acids, non-genomicnucleic acids, naturally occurring and not naturally occurring nucleicacids, and synthetic nucleic acids. This includes, by way ofnon-limiting example, nucleic acids associated with any organelle, suchas the mitochondria, ribosomal RNA, and nucleic acid molecules comprisedchimerically of one or more components that are not naturally occurringalong with naturally occurring components.

Additionally, a “nucleic acid molecule” may contain in part one or morenon-nucleotide-based components as exemplified by, but not limited to,amino acids and sugars. Thus, by way of example, but not limitation, aribozyme that is in part nucleotide-based and in part protein-based isconsidered a “nucleic acid molecule”.

In addition, by way of example, but not limitation, a nucleic acidmolecule that is labeled with a detectable moiety, such as a radioactiveor alternatively a non-radioactive label, is likewise considered a“nucleic acid molecule”.

The terms “nucleic acid sequence coding for” or a “DNA coding sequenceof” or a “nucleotide sequence encoding” a particular protein—as well asother synonymous terms—refer to a DNA sequence which is transcribed andtranslated into a protein when placed under the control of appropriateregulatory sequences. A “promotor sequence” is a DNA regulatory regioncapable of binding RNA polymerase in a cell and initiating transcriptionof a downstream (3′ direction) coding sequence. The promoter is part ofthe DNA sequence. This sequence region has a start codon at its 3′terminus. The promoter sequence does include the minimum number of baseswhere elements necessary to initiate transcription at levels detectableabove background. However, after the RNA polymerase binds the sequenceand transcription is initiated at the start codon (3′ terminus with apromoter), transcription proceeds downstream in the 3′ direction. Withinthe promotor sequence will be found a transcription initiation site(conveniently defined by mapping with nuclease S1) as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

The terms “nucleic acid encoding an protein” or “DNA encoding anprotein” or “polynucleotide encoding an protein” and other synonymousterms encompasses a polynucleotide which includes only coding sequencefor the protein as well as a polynucleotide which includes additionalcoding and/or non-Cq3 coding sequence.

In one preferred embodiment, a “specific nucleic acid molecule species”is defined by its chemical structure, as exemplified by, but not limitedto, its primary sequence. In another preferred embodiment, a specific“nucleic acid molecule species” is defined by a function of the nucleicacid species or by a function of a product derived from the nucleic acidspecies. Thus, by way of non-limiting example, a “specific nucleic acidmolecule species” may be defined by one or more activities or propertiesattributable to it, including activities or properties attributable itsexpressed product.

The instant definition of “assembling a working nucleic acid sample intoa nucleic acid library” includes the process of incorporating a nucleicacid sample into a vector-based collection, such as by ligation into avector and transformation of a host. A description of relevant vectors,hosts, and other reagents as well as specific non-limiting examplesthereof are provided hereinafter. The instant definition of “assemblinga working nucleic acid sample into a nucleic acid library” also includesthe process of incorporating a nucleic acid sample into anon-vector-based collection, such as by ligation to adaptors. Preferablythe adaptors can anneal to PCR primers to facilitate amplification byPCR.

Accordingly, in a non-limiting embodiment, a “nucleic acid library” iscomprised of a vector-based collection of one or more nucleic acidmolecules. In another preferred embodiment a “nucleic acid library” iscomprised of a non-vector-based collection of nucleic acid molecules. Inyet another preferred embodiment a “nucleic acid library” is comprisedof a combined collection of nucleic acid molecules that is in partvector-based and in part non-vector-based. Preferably, the collection ofmolecules comprising a library is searchable and separable according toindividual nucleic acid molecule species.

The present invention provides a “nucleic acid construct” oralternatively a “nucleotide construct” or alternatively a “DNAconstruct”. The term “construct” is used herein to describe a molecule,such as a polynucleotide (e.g., a phytase polynucleotide) may optionallybe chemically bonded to one or more additional molecular moieties, suchas a vector, or parts of a vector. In a specific—but by no meanslimiting—aspect, a nucleotide construct is exemplified by a DNAexpression DNA expression constructs suitable for the transformation ofa host cell.

An “oligonucleotide” (or synonymously an “oligo”) refers to either asingle stranded polydeoxynucleotide or two complementarypolydeoxynucleotide strands which may be chemically synthesized. Suchsynthetic oligonucleotides may or may not have a 5′ phosphate. Thosethat do not will not ligate to another oligonucleotide without adding aphosphate with an ATP in the presence of a kinase. A syntheticoligonucleotide will ligate to a fragment that has not beendephosphorylated. To achieve polymerase-based amplification (such aswith PCR), a “32-fold degenerate oligonucleotide that is comprised of,in series, at least a first homologous sequence, a degenerate N,N,G/Tsequence, and a second homologous sequence” is mentioned. As used inthis context, “homologous” is in reference to homology between the oligoand the parental polynucleotide that is subjected to thepolymerase-based amplification.

As used herein, the term “operably linked” refers to a linkage ofpolynucleotide elements in a functional relationship. A nucleic acid is“operably linked” when it is placed into a functional relationship withanother nucleic acid sequence. For instance, a promoter or enhancer isoperably linked to a coding sequence if it affects the transcription ofthe coding sequence. Operably linked means that the DNA sequences beinglinked are typically contiguous and, where necessary to join two proteincoding regions, contiguous and in reading frame.

A coding sequence is “operably linked to” another coding sequence whenRNA polymerase will transcribe the two coding sequences into a singlemRNA, which is then translated into a single polypeptide having aminoacids derived from both coding sequences. The coding sequences need notbe contiguous to one another so long as the expressed sequences areultimately processed to produce the desired protein.

As used herein the term “physiological conditions” refers totemperature, pH, ionic strength, viscosity, and like biochemicalparameters which are compatible with a viable organism, and/or whichtypically exist intracellularly in a viable cultured yeast cell ormammalian cell. For example, the intracellular conditions in a yeastcell grown under typical laboratory culture conditions are physiologicalconditions. Suitable in vitro reaction conditions for in vitrotranscription cocktails are generally physiological conditions. Ingeneral, in vitro physiological conditions comprise 50-200 mM NaCl orKCl, pH 6.5-8.5, 20-45° C. and 0.001-10 mM divalent cation (e.g., Mg++,Ca++); preferably about 150 mM NaCl or KCl, pH 7.2-7.6, 5 mM divalentcation, and often include 0.01-1.0 percent nonspecific protein (e.g.,BSA). A non-ionic detergent (Tween, NP-40, Triton X-100) can often bepresent, usually at about 0.001 to 2%, typically 0.05-0.2% (v/v).Particular aqueous conditions may be selected by the practitioneraccording to conventional methods. For general guidance, the followingbuffered aqueous conditions may be applicable: 10-250 mM NaCl, 5-50 mMTris HCl, pH 5-8, with optional addition of divalent cation(s) and/ormetal chelators and/or non-ionic detergents and/or membrane fractionsand/or anti-foam agents and/or scintillants.

The term “population” as used herein means a collection of componentssuch as polynucleotides, portions or polynucleotides or proteins. A“mixed population: means a collection of components which belong to thesame family of nucleic acids or proteins (i.e., are related) but whichdiffer in their sequence (i.e., are not identical) and hence in theirbiological activity.

A molecule having a “pro-form” refers to a molecule that undergoes anycombination of one or more covalent and noncovalent chemicalmodifications (e.g., glycosylation, proteolytic cleavage, dimerizationor oligomerization, temperature-induced or pH-induced conformationalchange, association with a co-factor, etc.) en route to attain a moremature molecular form having a property difference (e.g. an increase inactivity) in comparison with the reference pro-form molecule. When twoor more chemical modification (e.g. two proteolytic cleavages, or aproteolytic cleavage and a deglycosylation) can be distinguished enroute to the production of a mature molecule, the reference precursormolecule may be termed a “pre-pro-form” molecule.

A “property” can describe any characteristic, including a physical,chemical, or activity characteristic property of a protein or antibodyto be optimized. For example, in certain aspects, the predeterminedproperty, characteristic or activity to be optimized can be selectedfrom is selected from reduction of protein-protein aggregation,enhancement of protein stability, increased protein solubility,increased protein pH stability, increased protein temperature stability,increased protein solvent stability, increased selectivity, decreasedselectivity, introduction of glycosylation sites, introduction ofconjugation sites, reduction of immunogenicity, enhancement of proteinexpression, increase in antigen affinity, decrease in antigen affinity,change in binding affinity, change in immunogenicity, change incatalytic activity, pH optimization, or enhancement of specificity.Other properties or characteristics to be optimized include antibodystability in vivo (e.g., serum half-lives) and/or in vitro (e.g.,shelf-life); melting temperature (Tm) of the antibody (e.g., asdetermined by differential scanning calorimetry (DSC) or other methodknown in the art), the pI of the antibody (e.g., as determinedIsoelectric focusing (IEF) or other methods known in the art);solubility; binding properties (e.g., antibody-antigen binding constantssuch as, Ka, Kd, K_(on), K_(off)), equilibrium dissociation constant(K_(D)); antibody solubility (e.g., solubility in a pharmaceuticallyacceptable carrier, diluent or excipient), effector function (e.g.,antibody dependent cell-mediated cytotoxicity (ADCC)); expression leveland production levels (e.g., the yield of an antibody from a cell).

An “optimized” property refers to a desirable change in a particularproperty in a mutant protein or antibody compared to a templateantibody. In one aspect, an optimized property refers to wherein theimprovement is between about 1% and 500%, relative to the templateantibody or is between about 2 fold and 1000 fold, relative to thetemplate antibody.

As used herein, the term “pseudorandom” refers to a set of sequencesthat have limited variability, such that, for example, the degree ofresidue variability at another position, but any pseudorandom positionis allowed some degree of residue variation, however circumscribed.

“Quasi-repeated units”, as used herein, refers to the repeats to bere-assorted and are by definition not identical. Indeed the method isproposed not only for practically identical encoding units produced bymutagenesis of the identical starting sequence, but also thereassortment of similar or related sequences which may divergesignificantly in some regions. Nevertheless, if the sequences containsufficient homologies to be reasserted by this approach, they can bereferred to as “quasi-repeated” units.

As used herein “random peptide library” refers to a set ofpolynucleotide sequences that encodes a set of random peptides, and tothe set of random peptides encoded by those polynucleotide sequences, aswell as the fusion proteins contain those random peptides.

As used herein, “random peptide sequence” refers to an amino acidsequence composed of two or more amino acid monomers and constructed bya stochastic or random process. A random peptide can include frameworkor scaffolding motifs, which may comprise invariant sequences.

As used herein, “receptor” refers to a molecule that has an affinity fora given ligand. Receptors can be naturally occurring or syntheticmolecules. Receptors can be employed in an unaltered state or asaggregates with other species. Receptors can be attached, covalently ornon-covalently, to a binding member, either directly or via a specificbinding substance. Examples of receptors include, but are not limitedto, antibodies, including monoclonal antibodies and antisera reactivewith specific antigenic determinants (such as on viruses, cells, orother materials), cell membrane receptors, complex carbohydrates andglycoproteins, enzymes, and hormone receptors.

“Recombinant” proteins refer to enzymes produced by recombinant DNAtechniques, i.e., produced from cells transformed by an exogenous DNAconstruct encoding the desired protein. “Synthetic” proteins are thoseprepared by chemical synthesis.

The term “related polynucleotides” means that regions or areas of thepolynucleotides are identical and regions or areas of thepolynucleotides are heterologous.

“Reductive reassortment”, as used herein, refers to the increase inmolecular diversity that is accrued through deletion (and/or insertion)events that are mediated by repeated sequences.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides: “reference sequence,” “comparisonwindow,” “sequence identity,” “percentage of sequence identity,” and“substantial identity.”

A “reference sequence” is a defined sequence used as a basis for asequence comparison; a reference sequence may be a subset of a largersequence, for example, as a segment of a full-length cDNA or genesequence given in a sequence listing, or may comprise a complete cDNA orgene sequence. Generally, a reference sequence is at least 20nucleotides in length, frequently at least 25 nucleotides in length, andoften at least 50 nucleotides in length. Since two polynucleotides mayeach (1) comprise a sequence (i.e., a portion of the completepolynucleotide sequence) that is similar between the two polynucleotidesand (2) may further comprise a sequence that is divergent between thetwo polynucleotides, sequence comparisons between two (or more)polynucleotides are typically performed by comparing sequences of thetwo polynucleotides over a “comparison window” to identify and comparelocal regions of sequence similarity.

“Repetitive Index (RI)”, as used herein, is the average number of copiesof the quasi-repeated units contained in the cloning vector.

The term “saturation” refers to a technique of evolution wherein everypossible change is made at each position of a template polynucleotide ortemplate polypeptide; however the change at each position is notconfirmed by testing, but merely assumed statistically wherein themajority of possible changes or nearly every possible change isestimated to occur at each position of a template. Saturationmutagenesis refers to mutating the DNA of a region of a gene encoding aprotein that changes codon amino acid sequence of the protein and thenscreening the expressed mutants of essentially all of the mutants for animproved phenotype based on statistical over-sampling that approachescomprehensive coverage, but does not guarantee complete coverage.

The term “sequence identity” means that two polynucleotide sequences areidentical (i.e., on a nucleotide-by-nucleotide basis) over the window ofcomparison. The term “percentage of sequence identity” is calculated bycomparing two optimally aligned sequences over the window of comparison,determining the number of positions at which the identical nucleic acidbase (e.g., A, T, C, G, U, or I) occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison (i.e., thewindow size), and multiplying the result by 100 to yield the percentageof sequence identity. This “substantial identity”, as used herein,denotes a characteristic of a polynucleotide sequence, wherein thepolynucleotide comprises a sequence having at least 80 percent sequenceidentity, preferably at least 85 percent identity, often 90 to 95percent sequence identity, and most commonly at least 99 percentsequence identity as compared to a reference sequence of a comparisonwindow of at least 25-50 nucleotides, wherein the percentage of sequenceidentity is calculated by comparing the reference sequence to thepolynucleotide sequence which may include deletions or additions whichtotal 20 percent or less of the reference sequence over the window ofcomparison.

The term “silent mutation” refers to a codon change that does not resultin an amino acid change in an expressed polypeptide and is based onredundancy of codon usage for amino acid insertion.

As known in the art “similarity” between two proteins is determined bycomparing the amino acid sequence and its conserved amino acidsubstitutes of one protein to the sequence of a second protein.Similarity may be determined by procedures which are well-known in theart, for example, a BLAST program (Basic Local Alignment Search Tool atthe National Center for Biological Information).

As used herein, the term “single-chain antibody” refers to a polypeptidecomprising a VH domain and a VL domain in polypeptide linkage, generallyliked via a spacer peptide (e.g., [Gly-Gly-Gly-Gly-Ser]_(x)), and whichmay comprise additional amino acid sequences at the amino- and/orcarboxy-termini. For example, a single-chain antibody may comprise atether segment for linking to the encoding polynucleotide. As an examplea scFv is a single-chain antibody. Single-chain antibodies are generallyproteins consisting of one or more polypeptide segments of at least 10contiguous amino substantially encoded by genes of the immunoglobulinsuperfamily (e.g., see Williams and Barclay, 1989, pp. 361-368, which isincorporated herein by reference), most frequently encoded by a rodent,non-human primate, avian, porcine bovine, ovine, goat, or human heavychain or light chain gene sequence. A functional single-chain antibodygenerally contains a sufficient portion of an immunoglobulin superfamilygene product so as to retain the property of binding to a specifictarget molecule, typically a receptor or antigen (epitope).

The members of a pair of molecules (e.g., an antibody-antigen pair or anucleic acid pair) are said to “specifically bind” to each other if theybind to each other with greater affinity than to other, non-specificmolecules. For example, an antibody raised against an antigen to whichit binds more efficiently than to a non-specific protein can bedescribed as specifically binding to the antigen. (Similarly, a nucleicacid probe can be described as specifically binding to a nucleic acidtarget if it forms a specific duplex with the target by base pairinginteractions (see above).)

“Specific hybridization” is defined herein as the formation of hybridsbetween a first polynucleotide and a second polynucleotide (e.g., apolynucleotide having a distinct but substantially identical sequence tothe first polynucleotide), wherein substantially unrelatedpolynucleotide sequences do not form hybrids in the mixture.

The term “specific polynucleotide” means a polynucleotide having certainend points and having a certain nucleic acid sequence. Twopolynucleotides wherein one polynucleotide has the identical sequence asa portion of the second polynucleotide but different ends comprises twodifferent specific polynucleotides.

“Stringent hybridization conditions” means hybridization will occur onlyif there is at least 90% identity, preferably at least 95% identity andmost preferably at least 97% identity between the sequences. SeeSambrook et al., 1989, which is hereby incorporated by reference in itsentirety.

Also included in the invention are polypeptides having sequences thatare “substantially identical” to the sequence of a polypeptide, such asone of any SEQ ID NO disclosed herein. A “substantially identical” aminoacid sequence is a sequence that differs from a reference sequence onlyby conservative amino acid substitutions, for example, substitutions ofone amino acid for another of the same class (e.g., substitution of onehydrophobic amino acid, such as isoleucine, valine, leucine, ormethionine, for another, or substitution of one polar amino acid foranother, such as substitution of arginine for lysine, glutamic acid foraspartic acid, or glutamine for asparagine).

Additionally a “substantially identical” amino acid sequence is asequence that differs from a reference sequence or by one or morenon-conservative substitutions, deletions, or insertions, particularlywhen such a substitution occurs at a site that is not the active sitethe molecule, and provided that the polypeptide essentially retains itsbehavioural properties. For example, one or more amino acids can bedeleted from a phytase polypeptide, resulting in modification of thestructure of the polypeptide, without significantly altering itsbiological activity. For example, amino- or carboxyl-terminal aminoacids that are not required for phytase biological activity can beremoved. Such modifications can result in the development of smalleractive phytase polypeptides.

The present invention provides a “substantially pure protein”. The term“substantially pure protein” is used herein to describe a molecule, suchas a polypeptide (e.g., a phytase polypeptide, or a fragment thereof)that is substantially free of other proteins, lipids, carbohydrates,nucleic acids, and other biological materials with which it is naturallyassociated. For example, a substantially pure molecule, such as apolypeptide, can be at least 60%, by dry weight, the molecule ofinterest. The purity of the polypeptides can be determined usingstandard methods including, e.g., polyacrylamide gel electrophoresis(e.g., SDS-PAGE), column chromatography (e.g., high performance liquidchromatography (HPLC)), and amino-terminal amino acid sequence analysis.

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual macromolecular species in the composition),and preferably substantially purified fraction is a composition whereinthe object species comprises at least about 50 percent (on a molarbasis) of all macromolecular species present. Generally, a substantiallypure composition will comprise more than about 80 to 90 percent of allmacromolecular species present in the composition. Most preferably, theobject species is purified to essential homogeneity (contaminant speciescannot be detected in the composition by conventional detection methods)wherein the composition consists essentially of a single macromolecularspecies. Solvent species, small molecules (<500 Daltons), and elementalion species are not considered macromolecular species.

DETAILED DESCRIPTION OF THE INVENTION

The disclosure provides a method for generation of enhanced full lengthantibodies in mammalian cells. A library of humanized variants isprovided with high, validated framework diversity without requiringback-mutations to retain original affinity. No CDR grafting or phagedisplay is required. In one embodiment, humanized antibody libraries arescreened for antigen binding in ELISA compared to a donor antibody. Inanother embodiment, cell-based screening is used to determine biologicalactivity. In another embodiment, the method comprises isotype switchingcompared to the donor antibody.

Antibodies are a class of serum proteins which are induced followingcontact with an antigen. They bind specifically to the antigen whichinduced their formation. Male, Immunology, Gower Medical Publishing,London, 1986, pp. 19-34 Immunoglobulin (Ig) is a synonym for antibody.Structurally, antibody molecules have a general four polypeptide chainstructure consisting of two identical heavy chains (HC) and twoidentical light chains (LC), stabilized and cross-linked by intrachainand interchain disulfide bonds and non-covalent bonds. Heavy chains alsohave covalent carbohydrate portions. Different antibody classes consistof polymers of the four chain structure. Heavy chains are of 5 majortypes (γ, μ, δ, α, ε) and consist of 440-600 amino acid residues. Lightchains are of two major types (κ,λ) and have about 220-230 amino acidresidues. Both heavy and light chains are folded into domains.Proteolytic enzymes, such as papain and pepsin, can be used to split anantibody molecule into different characteristic fragments. Papainproduces two separate and identical Fab fragments, each with oneantigen-binding site, and one Fc fragment. Pepsin produces one F(ab′)₂fragment. Alberts et al., Molecular Biology of the Cell, 2nd ed., 1989,Garland Publishing, Inc.

Both light chains (LC) and heavy chains (HC) have a variable sequence attheir amino-terminal ends but a constant sequence at theircarboxyl-terminal ends. The light chains have a constant region about110 amino acids long and a variable region of the same size. The heavychains also have a variable region about 110 amino acids long, but theconstant region of the H chains is about 330 or 440 amino acid long,depending on the class of the H chain. Alberts et al., Molecular Biologyof the Cell, 2nd ed., 1989, Garland Publishing, Inc. at pp 1019. Onlypart of the variable region participates directly in the binding ofantigen. Studies have shown that the variability in the variable regionsof both L and H chains is for the most part restricted to three smallhypervariable regions (also called complementarity-determining regions,or CDRs) in each chain. The remaining parts of the variable region,known as framework regions (FR), are relatively constant. Alberts etal., Molecular Biology of the Cell, 2nd ed., 1989, Garland Publishing,Inc. at pp 1019-1020.

In a preferred embodiment, the methods of the disclosure provide fulllength humanized antibody molecules, not Fabs or partial lengthfragments. These antibody fragments, which retain some ability toselectively bind to an antigen (e.g., a polypeptide antigen) of theantibody from which they are derived, can be made using well knownmethods in the art (see, e.g., Harlow and Lane, supra), and aredescribed further, as follows. Antibodies can be used to isolatepreparative quantities of the antigen by immunoaffinity chromatography.Various other uses of such antibodies are to diagnose and/or stagedisease (e.g., neoplasia) and for therapeutic application to treatdisease, such as for example: neoplasia, autoimmune disease, AIDS,cardiovascular disease, infections, and the like. Chimeric, human-like,humanized or fully human antibodies are particularly useful foradministration to human patients.

Humanization by CDR Grafting Compared to Humanization Methods of theDisclosure

Humanization by CDR grafting, or reshaping, involves intercalating themouse CDRs from each immunoglobulin chain within the FW regions of ahuman variable region.

One method of CDR grafting can be used to create what is called termedframework-patched immunoglobulins and is disclosed in Leung et al., U.S.Pat. No. 7,321,026, which is incorporated herein by reference. Unlikeprevious described methods of humanization, which grafted CDRs from adonor onto the frameworks of a single acceptor immunoglobulin, segmentsof framework (FR1, FR2, FR3, and FR4), or FRs, were patched to replacethe corresponding FRs of the parent immunoglobulin. Free assortment ofthese FRs from different immunoglobulins and from different species wasmixed and matched into forming the final immunoglobulin chainImmunoglobulin chains were prepared utilizing one or morecomplementarity determining regions (CDR's) from a donor immunoglobulinand portions of framework sequences from one or more human, or primateimmunoglobulins. The individual FR sequences are selected by the besthomology between the non-human antibody and the human antibody template.This approach, however, is labor intensive, and the optimal frameworkregions are not be easily identified.

Another method of CDR grafting is described by Williams et al. inAntibody Engineering, Vol. 1, Chapter 21, Konterman and Dubel, (eds.),Springer-Verlag Berlin Heidelberg 2010, pp. 319. FR sequences areselected by the best homology between the non-human antibody and thehuman antibody template. Selection of the human variable regions isconsidered to be of critical importance. There are over 9,000 heavy andover 2,500 kappa antibodies in the public databases. These includeKabat, GenBank, and IMGT databases. By aligning these databases with theKabat numbering system and introducing gaps where necessary, each humanvariable region is scored for identity to the mouse sequence. Theresidue identity is determined at FW region, canonical, VH-VK interfaceresidues and residues are identified from the homology models ofpotential importance. In addition, N-glycosylation patterns in the FWregion are identified, which may lead to glycosylation-dependent effectson antibody binding. The resulting human variable region sequences arerefined by maximizing sequence identity and homology to the mouseantibody.

The typical CDR grafting strategy described by Williams et al. 2010starts with cloning and sequencing variable region cDNAs from a mouse Bcell hydridoma. Chimeric heavy and light chain constructs are preparedutilizing the cDNA sequences. CDR grafted human variable regions aredesigned in parallel and CDR grafted humanized heavy and light chainconstructs are prepared. Recombinant antibodies are expressed intransient transfection using chimeric and/or humanized expressionconstructs. The antigen binding potency of recombinant humanizedantibodies is tested. If potency is low, further humanized antibodyversions are prepared by substituting with selected framework mouseresidues. The goal is to obtain a humanized antibody with optimumantigen binding potency, but with minimum mouse framework regionantibodies. This process of humanization by CDR grafting is alsosomewhat labor intensive, potentially requiring multiple iterations toprepare a humanized antibody exhibiting the most desirablecharacteristics.

Another method of humanizing antibodies which also involves reshaping toreduce the immunogenicity involves synthesizing a combinatorial librarycomprising CDRs from a donor antibody fused in frame to frameworkregions from a sub-bank of framework regions. This technique, calledframework-shuffling of antibodies, is disclosed in Wu et al US2010/0216975, which is incorporated herein by reference. For example, Wuet al. prepared combinatorial sub-libraries that were assembledsequentially using the polymerase chain reaction (PCR) by overlapextension.

The disclosure provides a technique of express humanization ofantibodies with reduced immunogenicity; while maintaining or increasingantigen-binding specificity and affinity when compared to the donorantibody, and simultaneously optimizing protein expression. In oneaspect, no additional affinity maturation is required.

The disclosure provides a method of producing humanized antibodies froma template antibody in which the variable region or CDRs are derivedfrom the template antibody and the framework and constant regions of theantibody are derived from one or more human antibodies. In one aspect,the frameworks are from a human framework pool of functionally expressedhuman antibodies. In another aspect, a single sequence is utilized forframework region 4 in either or both of the light chain and the heavychain. In a further aspect, the sequence encoding framework 4 iscomprised in the expression vector. The variable region or CDRs derivedfrom the template antibody preferably have from about 90% to about 100%identity with the variable region or CDRs of the template antibody,although any and all modifications, including substitutions, insertionsand deletions, are contemplated so long as the humanized antibodymaintains the ability to bind to the target antigen.

The regions of the humanized antibodies that are derived from humanantibodies need not have 100% identity with the human antibodies. In apreferred embodiment, as many of the human amino acid residues aspossible are retained in order that immunogenicity is negligible, butthe human residues, in particular residues of the framework region, aresubstituted as required and as taught herein below in accordance withthe present invention. Such modifications as disclosed herein arenecessary to support the antigen binding site formed by the CDRs whilesimultaneously maximizing the humanization of the antibody. In onespecific aspect, the framework regions of the humanized antibodies thatare derived from the human framework pool have 100% identity with thehuman antibodies.

Each of the heavy and light chain variable regions contain three CDRsthat combine to form the antigen binding site. The three CDRs aresurrounded by four FR regions that primarily function to support theCDRs. The sequences of the CDRs within the sequences of the variableregions of the heavy and light chains can be identified bycomputer-assisted alignment according to Kabat et al. (1987) inSequences of Proteins of Immunological Interest, 4^(th) ed., UnitedStates Department of Health and Human Services, U.S. Government PrintingOffice, Washington, D.C., or by molecular modeling of the variableregions, for example utilizing the ENCAD program as described by Levitt(1983) J. Mol. Biol. 168:595.

In one embodiment the CDRs are derived from one or more templateantibodies. Determination of the heavy chain CDRs and light chain CDRsis well within the skill of one in the art. See, for example,http://www.bioinf.org.uk/abs/.

The sequences of the CDRs of the humanized antibody may be modified orevolved by any technique known in the art and may include insertions,substitutions and deletions to the extent that the humanized antibodymaintains the ability to bind to and the target antigen. The ordinarilyskilled artisan can ascertain the maintenance of this activity byperforming the functional assays described herein below.

The humanized HC variable domain encoding library derived from one ormore of the CDRs of the template antigen and humanized LC variabledomain encoding library derived from one or more of the CDRs of thetemplate antigen may be combined with the human constant and frameworkregions to form the humanized antibody. Human genes which encode theconstant (C) regions of the humanized antibodies, fragments and regionsof the present invention can be derived from a human fetal liverlibrary, by known methods. Human C region genes can be derived from anyhuman cell including those which express and produce humanimmunoglobulins. The human C_(H) region can be derived from any of theknown classes or isotypes of human H chains, including γ, μ, α, δ, ε,and subtypes thereof, such as G1, G2, G3 and G4. Since the H chainisotype is responsible for the various effector functions of anantibody, the choice of C_(H) region will be guided by the desiredeffector functions, such as complement fixation, or activity inantibody-dependent cellular cytotoxicity (ADCC). Preferably, the C_(H)region is derived from gamma 1 (IgG1).

The human C_(L) region can be derived from either human L chain isotype,kappa or lambda, preferably kappa.

Genes encoding human immunoglobulin C regions are obtained from humancells by standard cloning techniques (Sambrook, et al. (MolecularCloning: A Laboratory Manual, 2^(nd) Edition, Cold Spring Harbor Press,Cold Spring Harbor, N.Y. (1989) and Ausubel et al., eds. CurrentProtocols in Molecular Biology (1987-1993)). Human C region genes arereadily available from known clones containing genes representing thetwo classes of L chains, the five classes of H chains and subclassesthereof. Chimeric antibody fragments, such as F(ab¹)₂ and Fab, can beprepared by designing a chimeric H chain gene which is appropriatelytruncated. For example, a chimeric gene encoding an H chain portion ofan F(ab¹)₂ fragment would include DNA sequences encoding the CH1 domainand hinge region of the H chain, followed by a translational stop codonto yield the truncated molecule.

Generally, in one example, humanized antibodies, fragments and regionsof the present invention are produced by cloning DNA segments encodingthe H and L chain antigen-binding regions comprising one or more CDRs ofthe template antibody, and joining these DNA segments to DNA segmentsincluding C_(H) and C_(L) regions, respectively, to produce full lengthchimeric immunoglobulin-encoding genes.

The sequences of the variable regions of the antibody may be modified byinsertions, substitutions and deletions to the extent that the chimericantibody maintains the ability to bind to and inhibit the targetantigen. The ordinarily skilled artisan can ascertain the maintenance ofthis activity by performing appropriate functional assays.

Methods for engineering or humanizing non-human or human antibodies canbe used and are well known in the art. Generally, a humanized orengineered antibody has one or more amino acid residues from a sourcewhich is non-human, e.g., but not limited to mouse, rat, rabbit,non-human primate or other mammal. These human amino acid residues areoften referred to as “import” residues, which are typically taken froman “import” variable, constant or other domain of a known humansequence. Known human Ig sequences are disclosed, e.g.,www.ncbi.nlm.nih.gov/entrez/query.fcgi; www.atcc.org/phage/hdb.html;www.sciquest.com/; www.abcam.com/;www.antibodyresource.com/onlinecomp.html;www.public.iastate.edu/.about.pedro/research_tools.html;www.mgen.uni-heidelberg.de/SD/IT/IT.html;www.whfreeman.com/immunology/CH05/kuby05.htm;www.library.thinkquest.org/12429/Immune/Antibody.html; www hhmiorg/grants/lectures/1996/vlab/;www.path.cam.ac.uld.about.mrc7/mikeimages.html;www.antibodyresource.com/; mcb.harvard.edu/BioLinks/Immunology.html,www.immunologylink.com/; pathbox.wustl.edu/.about.hcenter/index.html;www.biotech.ufl.edu/.about.hcl/; www.pebio.com/pa/340913/340913.html;www.nal.usda.gov/awic/pubs/antibody/;www.m.ehime-u.ac.jp/.about.yasuhito/Elisa.html;www.biodesign.com/table.asp; www.icnet.uk/axp/facs/davies/links.html;www.biotech.ufl.edu/.about.fccl/protocol.html;www.isac-net.org/sites_geo.html;aximtl.imt.uni-marburg.de/.about.rek/AEPStart.html;baserv.uci.kun.n1Labout.jraats/linksl.html;www.recab.uni-hd.de/immuno.bme.nwvu.edu/;www.mrc-cpe.cam.ac.uk/imt-doc/public/INTRO.html;www.ibt.unam.mx/vir/V_mice.html; imgt.cnusc.fr:8104/;www.biochem.ucLac.uld.about.martin/abs/index.html; antibody.bath.ac.uk/;abgen.cvm.tamu.edu/lab/wwwabgen.html;www.unizh.chLabout.honegger/AHOseminar/SlideOl.html;www.cryst.bbk.ac.uld.about.ubcg07s/; www.nimrmrc.ac.uk/CC/ccaewg/ccaewg.htm;www.path.cam.ac.uk/.about.mrc7/humanisation/TAHHP.html;www.ibt.unam.mx/vir/structure/stat_aim.html;www.biosci.missouri.edu/smithgp/index.html;www.cryst.bioc.cam.ac.uk/.about.fmolina/Web-pages/Pept/spottech.html;www.jerini.de/fr_products.htm; www.patents.ibm.con/ibm.html. Kabat etal. Sequences of Proteins of Immunological Interest, U.S. Dept. Health(1983), each entirely incorporated herein by reference.

Such imported sequences can be used to reduce immunogenicity or reduce,enhance or modify binding, affinity, on-rate, off-rate, avidity,specificity, half-life, or any other suitable characteristic, as knownin the art. Generally part or all of the non-human or human CDRsequences are maintained while the non-human sequences of the variableand constant regions are replaced with human or other amino acids.Antibodies can also optionally be humanized with retention of highaffinity for the antigen and other favorable biological properties. Toachieve this goal, humanized antibodies can be optionally prepared by aprocess of analysis of the parental sequences and various conceptualhumanized products using three-dimensional models of the parental andhumanized sequences. Three-dimensional immunoglobulin models arecommonly available and are familiar to those skilled in the art.Computer programs are available which illustrate and display probablethree-dimensional conformational structures of selected candidateimmunoglobulin sequences. Inspection of these displays permits analysisof the likely role of the residues in the functioning of the candidateimmunoglobulin sequence, i.e., the analysis of residues that influencethe ability of the candidate immunoglobulin to bind its antigen. In thisway, FR residues can be selected and combined from the consensus andimport sequences so that the desired antibody characteristic, such asincreased affinity for the target antigen(s), is achieved. In general,the CDR residues are directly and most substantially involved ininfluencing antigen binding. Humanization or engineering of antibodiesof the present invention can be performed using any known method, suchas but not limited to those described in, Winter (Jones et al., Nature321:522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen etal., Science 239:1534 (1988)), Sims et al., J. Immunol. 151: 2296(1993); Chothia and Lesk, J. Mol. Biol. 196:901 (1987), Carter et al.,Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992); Presta et al., J. Immunol.151:2623 (1993), U.S. Pat. Nos. 5,723,323, 5,976,862, 5,824,514,5,817,483, 5,814,476, 5,763,192, 5,723,323, 5,766,886, 5,714,352,6,204,023, 6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539;4,816,567, PCT/: US98/16280, US96/18978, US91/09630, US91/05939,US94/01234, GB89/01334, GB91/01134, GB92/01755; WO90/14443, WO90/14424,WO90/14430, EP 229246, each entirely incorporated herein by reference,included references cited therein.

The human constant region of the humanized antibody of the invention canbe of any class (IgG, IgA, IgM, IgE, IgD, etc.) or isotype and cancomprise a kappa or lambda light chain. In one embodiment, the humanconstant region comprises an IgG heavy chain or defined fragment, forexample, at least one of isotypes, IgG1, IgG2, IgG3 or IgG4. In anotherembodiment, the humanized human antibody comprises an IgG1 heavy chainand a IgG1 K light chain. The isolated humanized antibodies of thepresent invention comprise antibody amino acid sequences disclosedherein encoded by any suitable polynucleotide as well as. Preferably,the humanized antibody binds the target antibody and, thereby partiallyor substantially neutralizes at least one biological activity of theprotein.

In one aspect, the humanized antibody will comprise an antigen-bindingregion that comprises at least one human complementarity determiningregion (CDR1, CDR2 and CDR3) or variant of at least one heavy chainvariable region and at least one human complementarity determiningregion (CDR4, CDR5 and CDR6) or variant of at least one light chainvariable region, derived from the template antibody.

In a particular embodiment, the antibody or antigen-binding fragment canhave an antigen-binding region that comprises at least a portion of atleast one heavy chain CDR (i.e., CDR1, CDR2 and/or CDR3) having theamino acid sequence of the corresponding CDRs 1, 2 and/or 3. In anotherparticular embodiment, the antibody or antigen-binding portion orvariant can have an antigen-binding region that comprises at least aportion of at least one light chain CDR (i.e., CDR4, CDR5 and/or CDR6)having the amino acid sequence of the corresponding CDRs 4, 5 and/or 6.In one embodiment the three heavy chain CDRs and the three light chainCDRs of the antibody or antigen-binding fragment have the amino acidsequence of the corresponding CDR of the template antibody. Suchantibodies can be prepared by chemically joining together the variousportions (e.g., CDRs, framework) of the antibody using conventionaltechniques, by preparing and expressing a (i.e., one or more) nucleicacid molecule that encodes the antibody using conventional techniques ofrecombinant DNA technology or by using any other suitable method andusing any of the possible redundant codons that will result inexpression of a polypeptide of the invention.

In another embodiment, the disclosure provides a method of humanizationof antibodies that comprises expression of a full length antibody in aeukaryotic cell production host; the method comprising selecting atemplate antibody; evolving one or more CDR sequences from the templateantibody to produce one or more CDR fragment libraries; ligating the CDRfragment libraries with a human framework pool from functionallyexpressed antibodies, wherein a single sequence for each frameworkregion 4 is utilized from the pool; screening the variant antibodies forat least one predetermined property, characteristic or activity;selecting a variant humanized antibody from the set of mutant antibodiesbased upon reduction of immunogenicity, and affinity for the antigen,compared to the template antibody. In one aspect, one or more of thevariant humanized antibodies are optimized for at least one additionalpredetermined property, characteristic or activity compared to thetemplate antibody; such as expression level; and the antibodies areexpressed in the same eukaryotic cell production host as in the evolvingstep for any commercial scale. In one aspect, the humanized antibody isselected from the library of humanized antibodies based upon (1) reducedimmunogenicity compared to the template antibody; (2) optimization ofthe at least one predetermined antigen binding property, characteristicor activity compared to the template antibody; and (3) a high level ofexpression when compared to other humanized antibodies in the library.

In one embodiment, the method of the disclosure comprises selection of atemplate antibody that is directed to a specific antigen of interest.The template antibody may be an existing murine monoclonal antibody, ora chimeric antibody, or even an existing humanized antibody for whichone or more characteristics is desired to be improved or optimized.

In one aspect, the template antibody is cloned and sequenced to identifysequences for FW1, CDR1, FW2, CDR2, FW3, CDR3, and optionally FW4 of theimmunoglobulin variable portions of both the heavy chain and the lightchain.

Fragment libraries of ds DNA encoding variants of one or more of HCCDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 derived by anymethod of evolution from the template antibody are prepared by any meansknown in the art. Specifically, the double stranded DNA fragmentlibraries comprise complementarity determining region (CDR) fragmentencoding libraries including fragments encoding all or a portion of oneor more of HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3derived from the template antibody. In one aspect, the libraries forone, two, three, four, or five of the HC CDR1, HC CDR2, HC CDR3, LCCDR1, LC CDR2 and LC CDR3 may contain a single member comprising thesequence of the corresponding region on the template antibody.

In another aspect, the evolving step comprises an evolution technique.Any method of evolution is performed on the nucleic acid sequenceencoding the template antibody, or a fragment thereof, such as a CDRfragment, to prepare an antibody or fragment library. In one aspect, theevolution is performed by a method of comprehensive protein engineeringto ultimately produce the set of mutant antibodies. The method ofcomprehensive protein engineering can be selected, for example, from oneor a combination of Comprehensive Positional Evolution (CPE™),Comprehensive Protein Synthesis (CPS™) Flex Evolution, SynergyEvolution, Comprehensive Positional Insertion evolution (CPI™) orComprehensive Positional Deletion evolution (CPD™). These techniques aredisclosed in detail in PCT/US2010/42302, filed Jul. 16, 2010, andincorporated herein by reference. In another aspect, the mutantantibodies are expressed in the same mammalian system used to generatethe human antibody library.

In another embodiment, a template hybridoma or recombinant antibody isselected for a particular target antigen; evolution of one or more CDRsof the template antibody is performed to ultimately provide a set ofmutant antibodies which are screened, for example, by use of antibodycell surface display in a mammalian cell system; and manufacturing isperformed in the same mammalian cell system used for screening.

In another embodiment, the selected template hybridoma/recombinantantibody is humanized and screened in the manufacturing host, followedby production in the manufacturing host, wherein the step ofoptimization (evolution) is omitted altogether.

In other aspects of the present invention, downstream expressionoptimization in manufacturing hosts is performed by evolving the Fcregion of the antibody, silent codons in the antibody, and/or the vectorand/or host genes used in protein expression. In one aspect, an Fclibrary is generated by any evolutionary technique. In one specificaspect of expression optimization, comprehensive evolution is performedon Fc domain of an antibody to create a library of Fc mutants which canbe used to select an optimal partner for any Fv. Optimization isdesigned for rapid attachment of all Fc variants to each new Fv region.Alternatively, a subset of these Fcs can be used to attach to differentFvs. Each of these Fc variant/Fv combinations is screened as afull-length antibody expressed in mammalian cells (e.g. CHO,cost-effective media) for optimal expression. Further, CPS can beperformed to screen all theoretical permutations of up to 12 or more ofthese CPE hits in mammalian cells for expression improvement. Specificdesirable codon changes can also be selected to identify clones withincreased expression. Silent codons are identified and CPE is performedon these positions. This CPE library is screened to identify optimalexpression hits. Further, all theoretical permutations of up to 12 ormore CPE hits can be used in the CPS process to generate a new librarythat can be screened in mammalian cells for expression improvement. Thetop CPS silent mutation hits are used to customize protein for optimalexpression in a specific cell line and media. This provides opportunityfor biosimilar fine structure control.

Other areas for enhancement of expression include: optimization of thevector, including promoter, splice sites, 5′ and 3′ termini, flankingsequences, reduction of gene deletion and rearrangement, improvement ofhost cell gene activities, optimization of host glycosylating enzymes,and chromosome wide host cell mutagenesis and selection. It has beendemonstrated that 5′ amino acid sequences are important for enhancementof expression.

Evolution of Lead Candidates

In another aspect, any method of protein evolution can be employed forsimultaneous evolution of antibody performance and expressionoptimization. Optimization of protein performance can includeimprovement of various characteristics such as affinity, pharmacokineticcharacteristics, tissue targeting, protein-protein aggregation,addressing high assay variability and modifying other in vivocharacteristics.

Methods for evolving molecules, including template antibodies of thepresent invention, include stochastic and non-stochastic methods.Published methods include random and non-random mutagenesis approaches.Any of these approaches can be employed to further evolve properties ofthe humanized antibodies of the disclosure toward a desiredcharacteristic, such as better stability in different temperature or pHenvironments, or better expression in a host cell. Other potentiallydesirable properties, such as improved catalytic activity, improvedprotein stability in various conditions, improved selectivity and/orsolubility, and improved expression results by improvement ofcharacteristics such as reduced aggregation can be selected for inevolution experiments.

Evolution can be performed directly in a eukaryotic host, such as amammalian cell host or a yeast cell host, that will be used fordownstream production of the therapeutic protein. Candidates can beevolved for optimal expression in the same host used to screen and/orevolve and to manufacture. Expression optimization can be achieved byoptimization of vectors used (vector components, such as promoters,splice sites, 5′ and 3′ termini and flanking sequences), genemodification of host cells to reduce gene deletions and rearrangements,evolution of host cell gene activities by in vivo or in vitro methods ofevolving relevant genes, optimization of host glycosylating enzymes byevolution of relevant genes, and/or by chromosome wide host cellmutagenesis and selection strategies to select for cells with enhancedexpression capabilities. Host cells are further described herein.

Cell surface display expression and screening technology (for example,as defined above) can be employed to screen libraries of evolvedproteins for candidates to be manufactured.

Current methods in widespread use for creating alternative proteins froma starting molecule are oligonucleotide-directed mutagenesistechnologies, error-prone polymerase chain reactions and cassettemutagenesis, in which the specific region to be optimized is replacedwith a synthetically mutagenized oligonucleotide. In these cases, anumber of mutant sites are generated around certain sites in theoriginal sequence.

In oligonucleotide-directed mutagenesis, a short sequence is replacedwith a synthetically mutagenized oligonucleotide. Error-prone PCR useslow-fidelity polymerization conditions to introduce a low level of pointmutations randomly over a long sequence. In a mixture of fragments ofunknown sequence, error-prone PCR can be used to mutagenize the mixture.In cassette mutagenesis, a sequence block of a single template istypically replaced by a (partially) randomized sequence.

Chimeric genes have been made by joining 2 polynucleotide fragmentsusing compatible sticky ends generated by restriction enzyme(s), whereeach fragment is derived from a separate progenitor (or parental)molecule. Another example is the mutagenesis of a single codon position(i.e. to achieve a codon substitution, addition, or deletion) in aparental polynucleotide to generate a single progeny polynucleotideencoding for a single site-mutagenized polypeptide.

Further, in vivo site specific recombination systems have been utilizedto generate hybrids of genes, as well as random methods of in vivorecombination, and recombination between homologous but truncated geneson a plasmid. Mutagenesis has also been reported by overlappingextension and PCR.

Non-random methods have been used to achieve larger numbers of pointmutations and/or chimerizations, for example comprehensive or exhaustiveapproaches have been used to generate all the molecular species within aparticular grouping of mutations, for attributing functionality tospecific structural groups in a template molecule (e.g. a specificsingle amino acid position or a sequence comprised of two or more aminoacids positions), and for categorizing and comparing specific groupingof mutations. U.S. Pat. No. 7,033,781 entitled “Whole cell engineeringmy mutagenizing a substantial portion of a starting genome, combiningmutations, and optionally repeating” describes a method of evolving anorganism toward desired characteristics. U.S. Pat. No. 6,764,835entitled “Saturation mutagenesis in directed evolution” and U.S. Pat.No. 6,562,594 entitled “Synthetic ligation reassembly in directedevolution” describe methods of exhaustively evolving and screening fordesired characteristics of molecules. Any such methods can be used inthe method of the present invention.

There is a difference between previously known methods of “saturationmutagenesis” and techniques of “comprehensive” evolution preferredherein. Saturation mutagenesis refers to a technique of evolutionwherein every possible change is made at each position of a templatepolynucleotide or template polypeptide; however the change at eachposition is not confirmed by testing, but merely assumed statistically.Comprehensive evolution refers to a technique of evolution wherein everypossible change is made at each position of a template polynucleotide ortemplate polypeptide and the polynucleotide or polypeptide is tested toconfirm the intended change has been made.

In another embodiment, the CPE/EvoMap may be used to identify andexploit fully mutable sites. In one aspect, exploitation of multiplefully mutable sites is termed Flex Evolution and is used to maketargeted changes such as introduction of sites for glycosylation (e.g.codons for amino acids for N- or O-linked glycosylation; Asn withinconsensus sequence Asn-Aa-Ser-Thr or Ser/Thr) and chemical conjugation.Flex evolution may also be used in design of protease cleavage sites,introduction of tags for purification and/or detection, site-specificlabeling, and the like. Further, codon optimization of silent mutationsmay be utilized for improvement of protein expression. In thisembodiment, termed Flex Evolution, following protein expression, themutant polypeptide libraries produced are rescreened for at least onepredetermined property, characteristic or activity compared to thetemplate polypeptide. In one aspect, the predetermined property includesreduction of protein-protein aggregation, enhancement of proteinstability, or increased protein solubility. In one aspect, the mutantpolypeptide libraries are screened for two or more propertiessimultaneously. In another aspect, any eukaryotic expression systemwhich glycosylates may be used for the introduction of glycosylationsites, such as, for example, mammalian, plant, yeast, and insect celllines.

In the technique of Flex Evolution, evaluation of bioinformatics andprotein x-ray crystal structures of related proteins, or the templateprotein or polypeptide, is useful for template optimization. In oneaspect, selected sites are not at contact residues. In another aspect,selection of non-surface protein mutations allows for reducedimmunogenicity risk.

Applications of Flex Evolution include, bit are not limited to,reduction of protein-protein aggregation, improvement of proteinsolubility, optimization of pharmacokinetics via glycosylationlibraries, optimization of protein secondary and tertiary structure anddeimmunization of antigenic sites directly via either mutation sets orindirectly through glycosylation masking.

In one aspect of Flex Evolution, an EvoMap™ is utilized to identifyfully mutable sites, CPS generation is performed with insertion ofglycosylating residues to fully mutable sites (or silent mutations fortranslation effects), and screening of combinatorial glycosylatedlibrary is performed by analytical analysis (e.g. Mass Spectroscopyanalysis, Dynamic Light Scattering), immunogenicity reduction (bybioinformatics or assay), and/or pharmacokinetic analysis (e.g. inFoxn1nu mice).

In one aspect, Flex evolution may be used for deimmunization toeliminate immunogenicity while maintaining function. Flex Evolutiondeimmunization can be performed by masking immunogenicity withglycosylation, identifying human hypersomatic mutation spectra aminoacid substitutions that may eliminate immunogenicity while maintainingfunction, reduction of dose for evading immunogenicity potential, andminimization of non-surface amino acid residue changes. Further,immunogenicity databases and algorithms can be used to identify andreplace potential MHC binding epitopes. In one aspect, in silicomodification prediction is coupled with CPE/CPS data to generatevariants.

Reduced propensity to generate T-cell epitopes and/or deimmunization maybe measured by techniques known in the art. Preferably, deimmunizationof proteins may be tested in vitro by T cell proliferation assay. Inthis assay PBMCs from donors representing >80% of HLA-DR alleles in theworld are screened for proliferation in response to either wild type ordeimmunized peptides. Ideally cell proliferation is only detected uponloading of the antigen-presenting cells with wild type peptides.Additional assays for deimmunization include human in vitro PBMCre-stimulation assays (e.g. interferon gamma (TH1) or IL4 (TH2) ELISA.Alternatively, one may test deimmunization by expressing HLA-DRtetramers representing all haplotypes. In order to test if de-immunizedpeptides are presented on HLA-DR haplotypes, binding of e.g.fluorescence-labeled peptides on PBMCs can be measured. Measurement ofHLA Class I and Class II transgenic mice for responses to target antigen(e.g. interferon gamma or IL4). Alternatively epitope library screeningwith educated T cells (MHCI 9mer; MHCII 20mer) from PBMC and/ortransgenic mouse assays. Furthermore, deimmunization can be proven bydetermining whether antibodies against the deimmunized molecules havebeen generated after administration in patients.

In one aspect, the present invention discloses the utilization ofprotein engineering methods to develop silent mutation codon optimizedFc variants with improved expression in eukaryotic cells. A silentmutation is one in which the variation of the DNA sequence does notresult in a change in the amino acid sequence of the protein. In oneaspect, codon mutagenesis is performed in the constant region foroptimization of eukaryotic cell expression. A codon optimized Fc variantwith improved expression properties while retaining the capacity tomediate effector functions improves the production of therapeuticantibodies. In this aspect, for example, a constant region of anantibody molecule can be evolved for screening in different expressionhosts, for example, mammalian cell lines expression screening utilizingCHO, HEK293 and COS-7.

Comprehensive Positional Insertion Evolution

In one embodiment, the disclosure provides methods of identifying andmapping mutant polypeptides formed from, or based upon, a templateantibody. Using a linear peptide as a simple example, in a first step, aset of naturally occurring amino acid variants (or a subset thereof, oramino acid derivatives) for each codon from position 2 to n (ncorresponding to the number of residues in the polypeptide chain) isgenerated by a process referred to herein as Comprehensive PositionalInsertion (CPI™) evolution.

In CPI™, an amino acid is inserted after each amino acid throughout atemplate polypeptide one at a time to generate a set of lengthenedpolypeptides. CPI can be used to insert 1, 2, 3, 4, or up to 5 new sitesat a time. Each of the 20 amino acids is added at each new position, oneat a time, creating a set of 20 different molecules at each new positionadded in the template. In this case, position 1, which is methionine andinvariant, is skipped. This procedure is repeated for each polypeptidechain of the target molecule. A minimum set of amino acid mutationscontains only one codon for each of the 20 natural amino acids.

The present invention relates to methods of identifying and mappingmutant polypeptides formed from, or based upon, a template polypeptide.Typically, the polypeptide will comprise n amino acid residues, whereinthe method comprises (a) generating n−[20×(n−1)] separate polypeptides,wherein each polypeptide differs from the template polypeptide in thatit has inserted after each position in the template each of the 20 aminoacids one at a time; assaying each polypeptide for at least onepredetermined property, characteristic or activity; and (b) for eachmember identifying any change in said property, characteristic oractivity relative to the template polypeptide.

In one embodiment, one or more regions are selected for mutagenesis toadd one position at a time as described above. In such case, nrepresents a subset or region of the template polypeptide. For example,where the polypeptide is an antibody, the entire antibody or one or morecomplementarity determining regions (CDRs) of the antibody are subjectedto mutagenesis to add one position at a time in the template polypeptideafter each position.

The invention thus includes methods of mapping a set of mutantantibodies formed from a template antibody having at least one, andpreferably six, complementarity determining regions (CDRs), the CDRstogether comprising n amino acid residues, the method comprising (a)generating n+[20×(n−1)] separate antibodies, wherein each antibodydiffers from the template antibody in that has inserted a singlepredetermined position, one at a time, after each position in thetemplate sequence; (b) assaying each set for at least one predeterminedproperty, characteristic or activity; and (c) for each memberidentifying any change in a property, characteristic or activityrelative to the template polypeptide. For antibodies, the predeterminedproperty, characteristic or property may be binding affinity and/orimmunogenicity, for example.

In addition, provided are methods of producing a set of mutantantibodies formed from a template antibody having at least onecomplementarity determining region (CDR), the CDR comprising n aminoacid residues, the method comprising: (a) generating n+[20×(n−1)]separate antibodies, wherein each antibody differs from the templateantibody in that it has an extra amino acid added at a singlepredetermined position of the CDR. In another embodiment, the antibodycomprises six CDRs, and together the CDRs comprise n amino acidresidues.

In another embodiment, the new lengthened polypeptides described aboveare further mutated and mapped after screening to identify a change in aproperty, characteristic or activity relative to the shortenedpolypeptide. Typically, the lengthened polypeptide will comprise n aminoacid residues, wherein the method comprises (a) generating n (n−1 in thecase where the initial residue is methionine) separate sets ofpolypeptides, each set comprising member polypeptides having X number ofdifferent predetermined amino acid residues at a single predeterminedposition of the polypeptide; wherein each set of polypeptides differs inthe single predetermined position; assaying each set for at least onepredetermined property, characteristic or activity; (b) for each memberidentifying any change in said property, characteristic or activityrelative to the template polypeptide; and optionally (c) creating afunctional map reflecting such changes. Preferably, the number ofdifferent member polypeptides generated is equivalent to n×X (or[n−1]×X, as the case may be).

In the alternative, the method comprises generating a single populationcomprising the sets of mutated polypeptides from the lengthenedpolypeptides. In this embodiment, the entire new population is screened,the individual members identified, and the functional map generated.

Typically, where each naturally occurring amino acid is used, X will be19 (representing the 20 naturally occurring amino acid residues andexcluding the particular residue present in a given position of thetemplate polypeptide). However, any subset of amino acids may be usedthroughout, and each set of polypeptides may be substituted with all ora subset of the total X used for the entire population.

However, it is recognized that each expression system may suffer fromcodon bias, in which insufficient tRNA pools can lead to translationstalling, premature translation termination, translation frameshiftingand amino acid misincorporation. Therefore, for expression optimizationeach set contains up to 63 different codons.

Each amino acid set is then screened for at least one, and preferablytwo or more, desirable characteristic such as improved function; neutralmutations, inhibitory mutations, and expression.

In one aspect, the lengthened polypeptides can be mapped to identify achange in a property, characteristic or activity resulting in theshortened polypeptides relative to the “wildtype”. The data for each setare combined for the entire polypeptide, or “target molecule”. Hits fromthe screening of the lengthened polypeptides (target molecules) can thenbe used for further comprehensive mutagenesis chain(s) and screening asdescribed herein. The data from mutagenesis provides a detailedfunctional map (referred to herein as an EvoMap™) of the target moleculeis generated. This map contains detailed information how each mutationaffects the performance/expression of the target molecule. It allows forthe identification of all sites where no changes can be made without aloss in protein function (or antigen/receptor binding in case ofantibodies). It also shows where changes can be made without affectingfunction.

In another aspect, CPE can be used to generate a library of 5, 10, up to15, or up to all 19 amino acids at each position of interest.

Comprehensive Positional Deletion Evolution

Comprehensive Positional Deletion Evolution (CPD™) relates to methods ofidentifying and mapping mutant polypeptides formed from, or based upon,a template polypeptide. CPD evolution deletes every amino acid throughthe protein one position at a time. Typically, the polypeptide willcomprise n amino acid residues, wherein the method comprises (a)generating n−1 (n−2 in the case where the initial residue is methionine)separate polypeptides, wherein each polypeptide differs from thetemplate polypeptide in that it lacks a single predetermined position;assaying each polypeptide for at least one predetermined property,characteristic or activity; and (b) for each member identifying anychange in said property, characteristic or activity relative to thetemplate polypeptide.

In one embodiment of CPD evolution, one or more regions are selected formutagenesis to remove one position at a time. In such case, n representsa subset or region of the template polypeptide. For example, where thepolypeptide is an antibody, the entire antibody or one or morecomplementarity determining regions (CDRs) of the antibody are subjectedto mutagenesis to remove one position at a time in the templatepolypeptide.

In one embodiment, CPD thus includes methods of mapping a set of mutantantibodies formed from a template antibody having at least one, andpreferably six, complementarity determining regions (CDRs), the CDRstogether comprising n amino acid residues, the method comprising (a)generating (n−1) separate antibodies, wherein each antibody differs fromthe template antibody in that lacks a single predetermined position; (b)assaying each set for at least one predetermined property,characteristic or activity; and (c) for each member identifying anychange in a property, characteristic or activity relative to thetemplate polypeptide. For antibodies, the predetermined property,characteristic or property may be binding affinity and/orimmunogenicity, for example.

One aspect of CPD evolution includes methods of producing a set ofmutant antibodies formed from a template antibody having at least onecomplementarity determining region (CDR), the CDR comprising n aminoacid residues, the method comprising: (a) generating n−1 separateantibodies, wherein each antibody differs from the template antibody inthat lacks a single predetermined position of the CDR. In anotherembodiment, the antibody comprises six CDRs, and together the CDRscomprise n amino acid residues.

In another embodiment of CPD evolution, the new shortened polypeptidesdescribed above are further mutated and mapped after screening toidentify a change in a property, characteristic or activity relative tothe shortened polypeptide. Typically, the shortened polypeptide willcomprise n amino acid residues, wherein the method comprises (a)generating n (n−1 in the case where the initial residue is methionine)separate sets of polypeptides, each set comprising member polypeptideshaving X number of different predetermined amino acid residues at asingle predetermined position of the polypeptide; wherein each set ofpolypeptides differs in the single predetermined position; assaying eachset for at least one predetermined property, characteristic or activity;(b) for each member identifying any change in said property,characteristic or activity relative to the template polypeptide; and (c)creating a functional map reflecting such changes. Preferably, thenumber of different member polypeptides generated is equivalent to n×X(or [n−1]×X, as the case may be).

In the alternative, the CPD method comprises generating a singlepopulation comprising the sets of mutated polypeptides from theshortened polypeptides. In this embodiment, the entire new population isscreened, the individual members identified, and the functional mapgenerated. Typically, where each naturally occurring amino acid is used,X will be 19 (representing the 20 naturally occurring amino acidresidues and excluding the particular residue present in a givenposition of the template polypeptide). However, any subset of aminoacids may be used throughout, and each set of polypeptides may besubstituted with all or a subset of the total X used for the entirepopulation.

Any mutational or synthetic means may be used to generate the set ofmutants in CPD evolution. In one embodiment, the generation ofpolypeptides comprises (i) subjecting a codon-containing polynucleotideencoding for the template polypeptide to polymerase-based amplificationusing a 64-fold degenerate oligonucleotide for each codon to bemutagenized, wherein each of the 64-fold degenerate oligonucleotides iscomprised of a first homologous sequence and a degenerate N,N,N tripletsequence, so as to generate a set of progeny polynucleotides; and (ii)subjecting the set of progeny polynucleotides to clonal amplificationsuch that polypeptides encoded by the progeny polynucleotides areexpressed.

In one embodiment of CPD evolution, the entire shortened polypeptide issubjected to saturation mutagenesis. In another embodiment, one or moreregions are selected for saturation mutagenesis. In such case, nrepresents a subset or region of the template polypeptide. For example,where the polypeptide is an antibody, the entire antibody or one or morecomplementarity determining regions (CDRs) of the antibody are subjectedto saturation mutagenesis.

The CPD evolution disclosure thus includes methods of mapping a set ofmutant antibodies formed from a shortened template antibody having atleast one, and preferably six, complementarity determining regions(CDRs), the CDRs together comprising n amino acid residues, the methodcomprising (a) generating n separate sets of antibodies, each setcomprising member antibodies having X number of different predeterminedamino acid residues at a single predetermined position of the CDR;wherein each set of antibodies differs in the single predeterminedposition; and the number of different member antibodies generated isequivalent to n×X; (b) assaying each set for at least one predeterminedproperty, characteristic or activity; (c) for each member identifyingany change in a property, characteristic or activity relative to thetemplate polypeptide; and (d) creating a structural positional map ofsuch changes. For antibodies, the predetermined property, characteristicor property may be binding affinity and/or immunogenicity. As set forthabove, in the alternative a single population comprising all sets ofmutated antibodies may be generated.

In addition, provided are methods of producing a set of mutantantibodies formed from a shortened template antibody having at least onecomplementarity determining region (CDR), the CDR comprising n aminoacid residues, the method comprising: (a) generating n separate sets ofantibodies, each set comprising member antibodies having X number ofdifferent predetermined amino acid residues at a single predeterminedposition of the CDR; wherein each set of antibodies differs in thesingle predetermined position; and the number of different memberantibodies generated is equivalent to n×X. In another embodiment,antibody comprises six CDRs, and together the CDRs comprise n amino acidresidues.

Combinatorial Protein Synthesis

Combinatorial Protein Synthesis (CPS™) involves combining individualhits from any evolutionary technique to combine two or more mutations.

Nucleic Acid Molecules

Using the information provided herein, such as the nucleotide sequencesencoding at least 70-100% of at least one of the contiguous CDR aminoacid sequences from the template antibody or specified fragments,variants or consensus sequences thereof, or a deposited vectorcomprising at least one of these sequences, a nucleic acid molecule ofthe present invention encoding at least one anti-antigen antibody can beobtained using methods described herein or as known in the art.

Nucleic acid molecules of the present invention can be in the form ofRNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA,including, but not limited to, cDNA and genomic DNA obtained by cloningor produced synthetically, or any combinations thereof. The DNA can betriple-stranded, double-stranded or single-stranded, or any combinationthereof. In one embodiment, the DNA is double stranded. Any portion ofat least one strand of the DNA or RNA can be the coding strand, alsoknown as the sense strand, or it can be the non-coding strand, alsoreferred to as the anti-sense strand.

Isolated nucleic acid molecules of the present invention can includenucleic acid molecules comprising an open reading frame (ORF),optionally with one or more introns, e.g., but not limited to, at leastone specified portion of at least one CDR, as CDR1, CDR2 and/or CDR3 ofat least one heavy chain or light chain; nucleic acid moleculescomprising the coding sequence for a template antibody or variableregion and nucleic acid molecules which comprise a nucleotide sequenceencoding a variant of the template antibody. Of course, the genetic codeis well known in the art. Thus, it would be routine for one skilled inthe art to generate such degenerate nucleic acid variants that code forspecific anti-antigen, for example, humanized antibodies of the presentinvention. See, e.g., Ausubel, et al., supra, and such nucleic acidvariants are included in the present invention. Non-limiting examples ofisolated nucleic acid molecules of the present invention include nucleicacid fragments encoding, respectively, all or a portion of HC CDR1, HCCDR2, HC CDR3, LC CDR1, LC CDR2, LC CDR3, HC variable region and LCvariable regions.

As indicated herein, nucleic acid molecules prepared by the methods ofthe disclosure which comprise a nucleic acid encoding a variant of atemplate antibody can include, but are not limited to, those encodingthe amino acid sequence of an antibody fragment, by itself, the codingsequence for the entire antibody or a portion thereof, the codingsequence for an antibody, fragment or portion, as well as additionalsequences, such as the coding sequence of at least one signal leader orfusion peptide, with or without the aforementioned additional codingsequences, such as at least one intron, together with additional,non-coding sequences, including but not limited to, non-coding 5′ and 3′sequences, such as the transcribed, non-translated sequences that play arole in transcription, mRNA processing, including splicing andpolyadenylation signals (for example—ribosome binding and stability ofmRNA); an additional coding sequence that codes for additional aminoacids, such as those that provide additional functionalities. Thus, thesequence encoding an antibody can be fused to a marker sequence, such asa sequence encoding a peptide that facilitates purification of the fusedantibody comprising an antibody fragment or portion.

Polynucleotides which Selectively Hybridize to a Polynucleotide asDescribed Herein

The present invention provides isolated nucleic acids that hybridizeunder selective hybridization conditions to a polynucleotide disclosedherein. Thus, the polynucleotides of this embodiment can be used forisolating, detecting, and/or quantifying nucleic acids comprising suchpolynucleotides. For example, polynucleotides of the present inventioncan be used to identify, isolate, or amplify partial or full-lengthclones in a deposited library. In some embodiments, the polynucleotidesare genomic or cDNA sequences isolated, or otherwise complementary to, acDNA from a human or mammalian nucleic acid library.

Preferably, the cDNA library comprises at least 80% full-lengthsequences, preferably at least 85% or 90% full-length sequences, andmore preferably at least 95% full-length sequences. The cDNA librariescan be normalized to increase the representation of rare sequences. Lowor moderate stringency hybridization conditions are typically, but notexclusively, employed with sequences having a reduced sequence identityrelative to complementary sequences. Moderate and high stringencyconditions can optionally be employed for sequences of greater identity.Low stringency conditions allow selective hybridization of sequenceshaving about 70% sequence identity and can be employed to identifyorthologous or paralogous sequences.

Prior methods of designed protein libraries include the technique ofGene Assembly Mutagenesis. Whole genes and plasmids can be assembledfrom relatively short, synthetic, overlapping oligodeoxyribonucleotides(oligos) by DNA polymerase extension. Gene Assembly mutagenesis achievesa population of gene variants assembled from short single strandedoligonucleotides encoding both strands of the gene and containingdegenerate bases at the targeted positions. Following assembly PCR thefull length gene variants are amplified using outside primers. Theassembly mutagenesis method has a technical limitation of introducingnon-targeted mutations at an elevated rate relative to routine PCR.While the extra diversity can be an advantage, it might be necessary toincrease the library size to ensure complete representation of allpossible intended sequences. See for example, Bassette et al., 2003,Construction of Designed Protein Libraries Using Gene AssemblyMutagenesis. Directed Evolution Library Creation, Methods and protocols.Edit by Frances H. Arnold and George Georgiou, Methods in MolecularBiology, 231, 29-37.

Optionally, polynucleotides of this invention will encode at least aportion of an antibody encoded by the polynucleotides described herein.The polynucleotides of this invention embrace nucleic acid sequencesthat can be employed for selective hybridization to a polynucleotideencoding an antibody of the present invention. See, e.g., Ausubel,supra; Colligan, supra, each entirely incorporated herein by reference.

Construction of Nucleic Acids

The isolated nucleic acids of the present invention can be made using(a) recombinant methods, (b) synthetic techniques, (c) purificationtechniques, or combinations thereof, as well-known in the art.

The nucleic acids can conveniently comprise sequences in addition to apolynucleotide of the present invention. For example, a multi-cloningsite comprising one or more endonuclease restriction sites can beinserted into the nucleic acid to aid in isolation of thepolynucleotide. Also, translatable sequences can be inserted to aid inthe isolation of the translated polynucleotide of the present invention.For example, a hexa-histidine marker sequence provides a convenientmeans to purify the proteins of the present invention. The nucleic acidof the present invention—excluding the coding sequence—is optionally avector, adapter, or linker for cloning and/or expression of apolynucleotide of the present invention.

Additional sequences can be added to such cloning and/or expressionsequences to optimize their function in cloning and/or expression, toaid in isolation of the polynucleotide, or to improve the introductionof the polynucleotide into a cell. Use of cloning vectors, expressionvectors, adapters, and linkers is well known in the art. (See, e.g.,Ausubel, supra; or Sambrook, supra).

Recombinant Methods for Constructing Nucleic Acids

The isolated nucleic acid compositions of this invention, such as RNA,cDNA, genomic DNA, or any combination thereof, can be obtained frombiological sources using any number of cloning methodologies known tothose of skill in the art. In some embodiments, oligonucleotide probesthat selectively hybridize, under stringent conditions, to thepolynucleotides of the present invention are used to identify thedesired sequence in a cDNA or genomic DNA library. The isolation of RNA,and construction of cDNA and genomic libraries, is well known to thoseof ordinary skill in the art. (See, e.g., Ausubel, supra; or Sambrook,supra).

Nucleic Acid Screening and Isolation Methods

A cDNA or genomic library can be screened using a probe based upon thesequence of a polynucleotide of the present invention, such as thosedisclosed herein. Probes can be used to hybridize with genomic DNA orcDNA sequences to isolate homologous genes in the same or differentorganisms. Those of skill in the art will appreciate that variousdegrees of stringency of hybridization can be employed in the assay; andeither the hybridization or the wash medium can be stringent. As theconditions for hybridization become more stringent, there must be agreater degree of complementarity between the probe and the target forduplex formation to occur. The degree of stringency can be controlled byone or more of temperature, ionic strength, pH and the presence of apartially denaturing solvent such as formamide. For example, thestringency of hybridization is conveniently varied by changing thepolarity of the reactant solution through, for example, manipulation ofthe concentration of formamide within the range of 0% to 50%. The degreeof complementarity (sequence identity) required for detectable bindingwill vary in accordance with the stringency of the hybridization mediumand/or wash medium. The degree of complementarity will optimally be100%, or 70-100%, or any range or value therein. However, it should beunderstood that minor sequence variations in the probes and primers canbe compensated for by reducing the stringency of the hybridizationand/or wash medium.

Methods of amplification of RNA or DNA are well known in the art and canbe used according to the present invention without undueexperimentation, based on the teaching and guidance presented herein.

Known methods of DNA or RNA amplification include, but are not limitedto, polymerase chain reaction (PCR) and related amplification processes(see, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188,to Mullis, et al.; 4,795,699 and 4,921,794 to Tabor, et al; U.S. Pat.No. 5,142,033 to Innis; U.S. Pat. No. 5,122,464 to Wilson, et al.; U.S.Pat. No. 5,091,310 to Innis; U.S. Pat. No. 5,066,584 to Gyllensten, etal; U.S. Pat. No. 4,889,818 to Gelfand, et al; U.S. Pat. No. 4,994,370to Silver, et al; U.S. Pat. No. 4,766,067 to Biswas; U.S. Pat. No.4,656,134 to Ringold) and RNA mediated amplification that usesanti-sense RNA to the target sequence as a template for double-strandedDNA synthesis (U.S. Pat. No. 5,130,238 to Malek, et al., with thetradename NASBA), the entire contents of which references areincorporated herein by reference. (See, e.g., Ausubel, supra; orSambrook, supra.)

For instance, polymerase chain reaction (PCR) technology can be used toamplify the sequences of polynucleotides of the present invention andrelated genes directly from genomic DNA or cDNA libraries. PCR and otherin vitro amplification methods can also be useful, for example, to clonenucleic acid sequences that code for proteins to be expressed, to makenucleic acids to use as probes for detecting the presence of the desiredmRNA in samples, for nucleic acid sequencing, or for other purposes.Examples of techniques sufficient to direct persons of skill through invitro amplification methods are found in Berger, supra, Sambrook, supra,and Ausubel, supra, as well as Mullis, et al., U.S. Pat. No. 4,683,202(1987); and Innis, et al., PCR Protocols A Guide to Methods andApplications, Eds., Academic Press Inc., San Diego, Calif. (1990).Commercially available kits for genomic PCR amplification are known inthe art. See, e.g., Advantage-GC Genomic PCR Kit (Clontech).Additionally, e.g., the T4 gene 32 protein (Boehringer Mannheim) can beused to improve yield of long PCR products.

Synthetic Methods for Constructing Nucleic Acids

The isolated nucleic acids of the present invention can also be preparedby direct chemical synthesis by known methods (see, e.g., Ausubel, etal., supra). Chemical synthesis generally produces a single-strandedoligonucleotide, which can be converted into double-stranded DNA byhybridization with a complementary sequence, or by polymerization with aDNA polymerase using the single strand as a template. One of skill inthe art will recognize that while chemical synthesis of DNA can belimited to sequences of about 100 or more bases, longer sequences can beobtained by the ligation of shorter sequences.

Recombinant Expression Cassettes

The present invention further provides recombinant expression cassettescomprising a nucleic acid of the present invention. A nucleic acidsequence of the present invention, for example a cDNA or a genomicsequence encoding an antibody of the present invention, can be used toconstruct a recombinant expression cassette that can be introduced intoat least one desired host cell. A recombinant expression cassette willtypically comprise a polynucleotide of the present invention operablylinked to transcriptional initiation regulatory sequences that willdirect the transcription of the polynucleotide in the intended hostcell. Both heterologous and non-heterologous (i.e., endogenous)promoters can be employed to direct expression of the nucleic acids ofthe present invention.

In some embodiments, isolated nucleic acids that serve as promoter,enhancer, or other elements can be introduced in the appropriateposition (upstream, downstream or in intron) of a non-heterologous formof a polynucleotide of the present invention so as to up or downregulate expression of a polynucleotide of the present invention. Forexample, endogenous promoters can be altered in vivo or in vitro bymutation, deletion and/or substitution.

Framework fragment encoding libraries of dsDNA which encode for all or aportion of HC FW1, HC FW2, HC FW3, LC FW1, LC FW2, and LC FW3 regionsare selected from a human framework pool selected only from functionallyexpressed antibodies from human germline sequences. The human frameworkpool is selected for maximum sequence diversity.

The method of the disclosure further comprises the step of assemblingfrom the HC fragment libraries by stepwise liquid phase ligation ofheavy chain FW encoding fragments and CDR encoding fragments in theorder of: FW1-CDR1-FW2-CDR2-FW3-CDR3 to produce a humanized HC variabledomain encoding library.

The method of the disclosure further comprises the step of assemblingfrom the LC fragment libraries by stepwise liquid phase ligation oflight chain FW encoding fragments and CDR encoding fragments in theorder of: FW1-CDR1-FW2-CDR2-FW3-CDR3 to produce a humanized LC variabledomain encoding library.

Liquid phase synthesis of combinatorial variable domain humanizedlibraries for the light chain and the heavy chain can be employed. Theassembly of a humanized light chain (LC) variable domain library, forexample, contains human light chain frameworks (FW) and non-humancomplementarity determining regions (CDR). The library is assembled by,for example, by using stepwise liquid phase ligation of FW and CDR DNAfragments. The libraries are assembled by using stepwise liquid phaseligation of FW and CDR DNA fragments in the order ofFW1-CDR1-FW2-CDR2-FW3-CDR3 by techniques known to one of skill in theart. For example, by the techniques of one or more of the followingreferences, each of which is incorporated herein by reference. Lo, B.K., 2003, Antibody humanization by CDR grafting. Antibody Engineering,Methods and protocols. Edit by Benny K. C. Lo, Methods in MolecularBiology, 248, 135-159; Kashmiri et al., 2003, Developing a minimallyimmunogenic humanized antibody by SDR grafting. Antibody Engineering,Methods and protocols. Edit by Benny K. C. Lo, Methods in MolecularBiology, 248, 361-376; Bassette, P. H., et al., 2003, Construction ofDesigned Protein Libraries Using Gene Assembly Mutagenesis. DirectedEvolution Library Creation, Methods and protocols. Edit. Arnold andGeorgiou, Methods in Molecular Biology, 231, 29-37; Chames, P., et al.,2001, Selections on Biotinylated antigens. Antibody Engineering, Edit byR. Kontermann and S. Dubel, Springer Lab Manual, 149-166; O'Brien S.,and Jones, T., 2001, Humanising antibodies by CDR grafting. AntibodyEngineering, Edit by R. Kontermann and S. Dubel, Springer Lab Manual,567-590. Assembly of fragments is further described in detail herein.

As stated, the invention also relates to a method of producing fulllength humanized antibodies, comprising one or more CDRs derived from atemplate antibody and framework regions from a human framework pool withframeworks only from functionally expresses antibodies. The humanframework pool is selected to provide maximum framework sequencediversity. Such humanized antibodies can include one or more amino acidsubstitutions, deletions or additions, either from natural mutations orhuman manipulation, as specified herein. Preferably, such antibodies canbind the antigen with high affinity (e.g., K_(D) less than or equal toabout 10⁻⁹ M) Amino acid sequences that are substantially the same asthe sequences described herein include sequences comprising conservativeamino acid substitutions, as well as amino acid deletions and/orinsertions. A conservative amino acid substitution refers to thereplacement of a first amino acid by a second amino acid that haschemical and/or physical properties (e.g., charge, structure, polarity,hydrophobicity/hydrophilicity) that are similar to those of the firstamino acid. Conservative substitutions include replacement of one aminoacid by another within the following groups: lysine (K), arginine (R)and histidine (H); aspartate (D) and glutamate (E); asparagine (N),glutamine (Q), serine (S), threonine (T), tyrosine (Y), K, R, H, D andE; alanine (A), valine (V), leucine (L), isoleucine (I), proline (P),phenylalanine (F), tryptophan (W), methionine (M), cysteine (C) andglycine (G); F, W and Y; C, S and T.

Of course, the number of amino acid substitutions a skilled artisanwould make depends on many factors, including those described above.Generally speaking, the number of amino acid substitutions, insertionsor deletions for any given humanized antibody, fragment or variant willnot be more than 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9,8, 7, 6, 5, 4, 3, 2, 1, such as 1-30 or any range or value therein, asspecified herein.

Amino acids in an anti-antigen antibody of the present invention thatare essential for function can be identified by methods known in theart, such as site-directed mutagenesis or alanine-scanning mutagenesis(e.g., Ausubel, supra, Chapters 8, 15; Cunningham and Wells, Science244:1081-1085 (1989)). The latter procedure introduces single alaninemutations at every residue in the molecule. The resulting mutantmolecules are then tested for biological activity, such as, but notlimited to at least one antigen neutralizing activity. Sites that arecritical for antibody binding can also be identified by structuralanalysis such as crystallization, nuclear magnetic resonance orphotoaffinity labeling (Smith, et al., J. Mol. Biol. 224:899-904 (1992)and de Vos, et al., Science 255:306-312 (1992)).

The method of the disclosure also comprises the step of cloning theassembled humanized heavy chain variable domain library and theassembled light chain variable domain library into an expression vectorto create a humanization library. In one aspect, the expression vectorcomprises a nucleotide sequence encoding framework region 4. Thehumanization library is transfected into cells.

Vectors and Host Cells

The present invention also relates to vectors that include isolatednucleic acid molecules of the present invention, host cells that aregenetically engineered with the recombinant vectors, and the productionof at least one humanized antibody by recombinant techniques, as is wellknown in the art. See, e.g., Sambrook, et al., supra; Ausubel, et al.,supra, each entirely incorporated herein by reference.

The polynucleotides can optionally be joined to a vector containing aselectable marker for propagation in a host. Generally, a plasmid vectoris introduced in a precipitate, such as a calcium phosphate precipitate,or in a complex with a charged lipid. If the vector is a virus, it canbe packaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

The DNA insert should be operatively linked to an appropriate promoter.The expression constructs will further contain sites for transcriptioninitiation, termination and, in the transcribed region, a ribosomebinding site for translation. The coding portion of the maturetranscripts expressed by the constructs will preferably include atranslation initiating at the beginning and a termination codon (e.g.,UAA, UGA or UAG) appropriately positioned at the end of the mRNA to betranslated, with UAA and UAG preferred for mammalian or eukaryotic cellexpression.

Expression vectors will preferably but optionally include at least oneselectable marker. Such markers include, e.g., but not limited to,methotrexate (MTX), dihydrofolate reductase (DHFR, U.S. Pat. Nos.4,399,216; 4,634,665; 4,656,134; 4,956,288; 5,149,636; 5,179,017,ampicillin, neomycin (G418), mycophenolic acid, or glutamine synthetase(GS, U.S. Pat. Nos. 5,122,464; 5,770,359; 5,827,739) resistance foreukaryotic cell culture, and tetracycline or ampicillin resistance genesfor culturing in E. coli and other bacteria or prokaryotics (the abovepatents are entirely incorporated hereby by reference). Appropriateculture mediums and conditions for the above-described host cells areknown in the art. Suitable vectors will be readily apparent to theskilled artisan. Introduction of a vector construct into a host cell canbe effected by calcium phosphate transfection, DEAE-dextran mediatedtransfection, cationic lipid-mediated transfection, electroporation,transduction, infection or other known methods. Such methods aredescribed in the art, such as Sambrook, supra, Chapters 1-4 and 16-18;Ausubel, supra, Chapters 1, 9, 13, 15, 16.

Cloning and Expression of humanized antibodies in Mammalian Cells

A typical mammalian expression vector contains at least one promoterelement, which mediates the initiation of transcription of mRNA, theantibody coding sequence, and signals required for the termination oftranscription and polyadenylation of the transcript. Additional elementsinclude enhancers, Kozak sequences and intervening sequences flanked bydonor and acceptor sites for RNA splicing. Highly efficienttranscription can be achieved with the early and late promoters fromSV40, the long terminal repeats (LTRS) from retroviruses, e.g., RSV,HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).However, cellular elements can also be used (e.g., the human actinpromoter). Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as pIRES1neo, pRetro-Off,pRetro-On, PLXSN, or pLNCX (Clonetech Labs, Palo Alto, Calif.), pcDNA3.1(+/−), pcDNA/Zeo (+/−) or pcDNA3.1/Hygro (+/−) (Invitrogen), PSVL andPMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC37146) and pBC12MI (ATCC 67109). Mammalian host cells that could be usedinclude human Hela 293, H9 and Jurkat cells, mouse NIH3T3 and C127cells, Cos 1, Cos 7 and CV 1, quail QC1-3 cells, mouse L cells andChinese hamster ovary (CHO) cells.

Alternatively, the gene can be expressed in stable cell lines thatcontain the gene integrated into a chromosome. The co-transfection witha selectable marker such as dhfr, gpt, neomycin, or hygromycin allowsthe identification and isolation of the transfected cells.

The transfected gene can also be amplified to express large amounts ofthe encoded antibody. The DHFR (dihydrofolate reductase) marker isuseful to develop cell lines that carry several hundred or even severalthousand copies of the gene of interest. Another useful selection markeris the enzyme glutamine synthase (GS) (Murphy, et al., Biochem. J.227:277-279 (1991); Bebbington, et al., Bio/Technology 10:169-175(1992)). Using these markers, the mammalian cells are grown in selectivemedium and the cells with the highest resistance are selected. Thesecell lines contain the amplified gene(s) integrated into a chromosome.Chinese hamster ovary (CHO) and NSO cells are often used for theproduction of antibodies.

Cloning and Expression in CHO Cells

In one aspect, the isolated variable and constant region encoding DNAand the dephosphorylated vector are ligated with T4 DNA ligase. E. coliHB101 or XL-1 Blue cells are then transformed and bacteria areidentified that contain the fragment inserted into plasmid pC4 using,for instance, restriction enzyme analysis.

For example, in one aspect, Chinese hamster ovary (CHO) cells lacking anactive DHFR gene are used for transfection. 5 μg of the expressionplasmid pC4 is cotransfected with 0.5 μg of the plasmid pSV2-neo usinglipofectin. The plasmid pSV2neo contains a dominant selectable marker,the neo gene from Tn5 encoding an enzyme that confers resistance to agroup of antibiotics including G418. The cells are seeded in alpha minusMEM supplemented with 1 μg/ml G418. After 2 days, the cells aretrypsinized and seeded in hybridoma cloning plates (Greiner, Germany) inalpha minus MEM supplemented with 10, 25, or 50 ng/ml of methotrexateplus 1 μg/ml G418. After about 10-14 days single clones are trypsinizedand then seeded in 6-well petri dishes or 10 ml flasks using differentconcentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (1 mM, 2 mM, 5 mM, 10 mM, 20 mM). The same procedure isrepeated until clones are obtained that grow at a concentration of100-200 mM. Expression of the desired gene product is analyzed, forinstance, by SDS-PAGE and Western blot, ELISA, or by reverse phase HPLCanalysis.

Illustrative of cell cultures useful for the production of theantibodies, specified portions or variants thereof, are mammalian cells.Mammalian cell systems often will be in the form of monolayers of cellsalthough mammalian cell suspensions or bioreactors can also be used. Anumber of suitable host cell lines capable of expressing intactglycosylated proteins have been developed in the art. In one aspect, thecells are selected from a eukaryotic cell production host cell lineselected from a member of the group consisting of 3T3 mouse fibroblastcells; BHK21 Syrian hamster fibroblast cells; MDCK, dog epithelialcells; Hela human epithelial cells; PtK1 rat kangaroo epithelial cells;SP2/0 mouse plasma cells; and NS0 mouse plasma cells; HEK 293 humanembryonic kidney cells; COS monkey kidney cells, including COS-1 (e.g.,ATCC CRL 1650), COS-7 (e.g., ATCC CRL-1651); CHO, CHO-S Chinese hamsterovary cells; R1 mouse embryonic cells; E14.1 mouse embryonic cells; H1human embryonic cells; H9 human embryonic cells; PER C.6, humanembryonic cells; S. cerevisiae yeast cells; and picchia yeast cells. Inone specific aspect, the eukaryotic cell production host cell line isCHO-S. In another specific aspect, the eukaryotic cell production hostcell line is HEK293. In a further specific aspect, the eukaryotic cellproduction host cell line is CHOK1SV. In another specific aspect, theeukaryotic cell production host cell line is NS0.

Expression vectors for these cells can include one or more of thefollowing expression control sequences, such as, but not limited to anorigin of replication; a promoter (e.g., late or early SV40 promoters,the CMV promoter (U.S. Pat. Nos. 5,168,062; 5,385,839), an HSV tkpromoter, a pgk (phosphoglycerate kinase) promoter, an EF-1 alphapromoter (U.S. Pat. No. 5,266,491), at least one human immunoglobulinpromoter; an enhancer, and/or processing information sites, such asribosome binding sites, RNA splice sites, polyadenylation sites (e.g.,an SV40 large T Ag poly A addition site), and transcriptional terminatorsequences. See, e.g., Ausubel et al., supra; Sambrook, et al., supra.Other cells useful for production of nucleic acids or proteins of thepresent invention are known and/or available, for instance, from theAmerican Type Culture Collection Catalogue of Cell Lines and Hybridomas(www.atcc.org) or other known or commercial sources.

When eukaryotic host cells are employed, polyadenlyation ortranscription terminator sequences are typically incorporated into thevector. An example of a terminator sequence is the polyadenlyationsequence from the bovine growth hormone gene. Sequences for accuratesplicing of the transcript can also be included. An example of asplicing sequence is the VP1 intron from SV40 (Sprague, et al., J.Virol. 45:773-781 (1983)). Additionally, gene sequences to controlreplication in the host cell can be incorporated into the vector, asknown in the art.

Nucleic acids of the present invention can be expressed in a host cellby turning on (by manipulation) in a host cell that contains endogenousDNA encoding an antibody of the present invention. Such methods are wellknown in the art, e.g., as described in U.S. Pat. Nos. 5,580,734,5,641,670, 5,733,746, and 5,733,761, entirely incorporated herein byreference.

The method of the disclosure also comprises the step of expressing fulllength humanized antibodies in the cells to create a humanized antibodylibrary. In one aspect, the cell is a eukaryotic cell production hostwith antibody cell surface display. In another aspect, one or both ofthe screening steps is performed in the eukaryotic cell production host.

Production of an Antibody

At least one humanized antibody of the present invention can beoptionally produced by a cell line, a mixed cell line, an immortalizedcell or clonal population of immortalized cells, as well known in theart. See, e.g., Ausubel, et al., ed., Current Protocols in MolecularBiology, John Wiley & Sons, Inc., NY, N.Y. (1987-2001); Sambrook, etal., Molecular Cloning: A Laboratory Manual, 2.sup.nd Edition, ColdSpring Harbor, N.Y. (1989); Harlow and Lane, antibodies, a LaboratoryManual, Cold Spring Harbor, N.Y. (1989). Colligan, et al., eds., CurrentProtocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001);Colligan et al., Current Protocols in Protein Science, John Wiley &Sons, NY, N.Y., (1997-2001), each entirely incorporated herein byreference.

In one approach, a hybridoma is produced by fusing a suitable immortalcell line (e.g., a myeloma cell line such as, but not limited to, Sp2/0,Sp2/0-AG14, NSO, NS1, NS2, AE-1, L.5, >243, P3X63Ag8.653, Sp2 SA3, Sp2MAI, Sp2 SS1, Sp2 SAS, U937, MLA 144, ACT IV, MOLT4, DA-1, JURKAT, WEHI,K-562, COS, RAJI, NIH 3T3, HL-60, MLA 144, NAMAIWA, NEURO 2A), or thelike, or heteromylomas, fusion products thereof, or any cell or fusioncell derived therefrom, or any other suitable cell line as known in theart. See, e.g., www.atcc.org, www.lifetech.com., and the like, withantibody producing cells, such as, but not limited to, isolated orcloned spleen, peripheral blood, lymph, tonsil, or other immune or Bcell containing cells, or any other cells expressing heavy or lightchain constant or variable or framework or CDR sequences, either asendogenous or heterologous nucleic acid, as recombinant or endogenous,viral, bacterial, algal, prokaryotic, amphibian, insect, reptilian,fish, mammalian, rodent, equine, ovine, goat, sheep, primate,eukaryotic, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA,chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single, double or triplestranded, hybridized, and the like or any combination thereof. See,e.g., Ausubel, supra, and Colligan, Immunology, supra, chapter 2,entirely incorporated herein by reference.

Any other suitable host cell can also be used for expressingheterologous or endogenous nucleic acid encoding an antibody, specifiedfragment or variant thereof, of the present invention. The fused cells(hybridomas) or recombinant cells can be isolated using selectiveculture conditions or other suitable known methods, and cloned bylimiting dilution or cell sorting, or other known methods. Cells whichproduce antibodies with the desired specificity can be selected by asuitable assay (e.g., ELISA).

Antibodies of the present invention can also be prepared using at leastone humanized antibody encoding nucleic acid to provide transgenicanimals or mammals, such as goats, cows, horses, sheep, and the like,that produce such antibodies in their milk. Such animals can be providedusing known methods. See, e.g., but not limited to, U.S. Pat. Nos.5,827,690; 5,849,992; 4,873,316; 5,849,992; 5,994,616, 5,565,362;5,304,489, and the like, each of which is entirely incorporated hereinby reference.

Antibodies of the present invention can additionally be prepared usingat least one humanized antibody encoding nucleic acid to providetransgenic plants and cultured plant cells (e.g., but not limited totobacco and maize) that produce such antibodies, specified portions orvariants in the plant parts or in cells cultured therefrom. As anon-limiting example, transgenic tobacco leaves expressing recombinantproteins have been successfully used to provide large amounts ofrecombinant proteins, e.g., using an inducible promoter. See, e.g.,Cramer et al., Curr. Top. Microbol. Immunol. 240:95-118 (1999) andreferences cited therein. Also, transgenic maize have been used toexpress mammalian proteins at commercial production levels, withbiological activities equivalent to those produced in other recombinantsystems or purified from natural sources. See, e.g., Hood et al., Adv.Exp. Med. Biol. 464:127-147 (1999) and references cited therein.Antibodies have also been produced in large amounts from transgenicplant seeds including antibody fragments, such as single chainantibodies (scFv's), including tobacco seeds and potato tubers. See,e.g., Conrad et al., Plant Mol. Biol. 38:101-109 (1998) and referencescited therein. Thus, antibodies of the present invention can also beproduced using transgenic plants, according to known methods. See also,e.g., Fischer et al., Biotechnol. Appl. Biochem. 30:99-108 (October,1999), Ma et al., Trends Biotechnol. 13:522-7 (1995); Ma et al., PlantPhysiol. 109:341-6 (1995); Whitelam et al., Biochem Soc. Trans.22:940-944 (1994); and references cited therein. Each of the abovereferences is entirely incorporated herein by reference.

Purification of an Antibody

A humanized antibody can be recovered and purified from recombinant cellcultures by well-known methods including, but not limited to, protein Apurification, protein G purification, ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. High performance liquid chromatography (“HPLC”) can alsobe employed for purification. See, e.g., Colligan, Current Protocols inImmunology, or Current Protocols in Protein Science, John Wiley & Sons,NY, N.Y., (1997-2001), e.g., chapters 1, 4, 6, 8, 9, and 10, eachentirely incorporated herein by reference.

Antibodies of the present invention include naturally purified products,products of chemical synthetic procedures, and products produced byrecombinant techniques from a eukaryotic host, including, for example,yeast, higher plant, insect and mammalian cells. Depending upon the hostemployed in a recombinant production procedure, the antibody of thepresent invention can be glycosylated or can be non-glycosylated, withglycosylated preferred. Such methods are described in many standardlaboratory manuals, such as Sambrook, supra, Sections 17.37-17.42;Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20, Colligan, ProteinScience, supra, Chapters 12-14, all entirely incorporated herein byreference.

Purified antibodies can be characterized by, for example, ELISA,ELISPOT, flow cytometry, immunocytology, Biacore® analysis, SapidyneKinExA™ kinetic exclusion assay, SDS-PAGE and Western blot, or by HPLCanalysis as well as by a number of other functional assays disclosedherein.

The method of the disclosure also comprises the step of screening thehumanized antibody library to determine the expression level of thehumanized antibodies. The method of the disclosure also comprises thestep of screening the humanized antibody library to determine theaffinity of the humanized antibodies for the antigen compared to theaffinity of the template antibody to the antigen. In one aspect,purified antibodies are screened. In another aspect, the eukaryotic cellproduction host is capable of antibody cell surface display, and thescreening steps are performed in the eukaryotic cell production host.

In specific aspects, screening steps are selected from the groupconsisting of quantitative ELISA; affinity ELISA; ELISPOT; flowcytometry, immunocytology, Biacore® surface plasmon resonance analysis,Sapidyne KinExA™ kinetic exclusion assay; SDS-PAGE; Western blot, andHPLC analysis as well as by a number of other functional assaysdisclosed herein.

In other aspects of the present invention, downstream expressionoptimization in manufacturing hosts is performed by evolving the Fcregion of the antibody, silent codons in the antibody, and/or the vectorand/or host genes used in protein expression. In one aspect, an Fclibrary is generated by any evolutionary technique. In one specificaspect of expression optimization, CPE is performed on Fc domain of anantibody to create a library of Fc mutants which can be used to selectan optimal partner for any Fv. Optimization is designed for rapidattachment of all Fc CPE variants to each new Fv region. Alternatively,a subset of these Fcs can be used to attach to different Fvs. Each ofthese Fc CPE variant/Fv combinations is screened as a full-lengthantibody expressed in mammalian cells (e.g. CHO, cost-effective media)for optimal expression. Further, CPS can be performed to screen alltheoretical permutations of up to 12 or more of these CPE hits inmammalian cells for expression improvement. Specific desirable codonchanges can also be selected to identify clones with increasedexpression. Silent codons are identified and CPE is performed on thesepositions. This CPE library is screened to identify optimal expressionhits. Further, all theoretical permutations of up to 12 or more CPE hitscan be used in the CPS process to generate a new library that can bescreened in mammalian cells for expression improvement. The top CPSsilent mutation hits are used to customize protein for optimalexpression in a specific cell line and media. This provides opportunityfor biosimilar fine structure control.

Other areas for enhancement of expression include: optimization of thevector, including promoter, splice sites, 5′ and 3′ termini, flankingsequences, reduction of gene deletion and rearrangement, improvement ofhost cell gene activities, optimization of host glycosylating enzymes,and chromosome wide host cell mutagenesis and selection. It has beendemonstrated that 5′ amino acid sequences are important for enhancementof expression.

EXAMPLES Abbreviations

-   BSA—bovine serum albumin-   EIA—enzyme immunoassay-   FBS—fetal bovine serum-   H₂O₂—hydrogen peroxide-   HRP—horseradish peroxidase-   Ig—immunoglobulin-   IL-6-Interleukin-6-   IP—intraperitoneal-   IV—intravenous-   Mab—monoclonal antibody-   OD—optical density-   OPD—o-Phenylenediamine dihydrochloride-   PEG—polyethylene glycol-   PSA—penicillin, streptomycin, amphotericin-   RT—room temperature-   SQ—subcutaneous-   v/v—volume per volume-   w/v—weight per volume

Example 1 Example 1. Preparation of Heavy Chain and Light Chain DoubleStranded DNA Fragments

This protocol describes the preparation of double stranded (ds) DNAfragments which are used for the assembly of heavy chain and light chainvariable domains [[SOP 2A)]]. The dsDNA fragments are prepared first byannealing synthetic oligonucleotides (oligos). The Oligos are5′-phosphorylated to allow the fragments to ligate to each other andinto the cloning vector.

Reagents, consumables and equipment required for this procedure areshown in

TABLE 1 Table 1. Reagents, Consumables, and Equipment. DescriptionApproved Supplier Catalogue No. SeaKem LE agarose Cambrex 50004 Ethidiumbromide Sigma-Aldrich E1510 TAE buffer (10x) - 1000 ml Ambion 9869 1MTris/HCl, pH 8.0 - 100 ml Ambion 9855G 2M KCl - 100 ml Ambion 9640G DTT(powder form) Fisher BP1725 1M MgCl2 - 100 ml Ambion 9530G Nuclease freewater - 5 × 100 ml Ambion 9939 Agarose gel apparatus Bio-Rad 1707764Agarose gel power supply Bio-Rad 1645050 4% agarose gel See Appendix 1N/A Agarose gel loading buffer Invitrogen 10816-015 25 base pair (bp)DNA ladder Invitrogen 10597-011 Microcentrifuge Eppendorf 5417C Highspeed centrifuge Beckman Avanti J-30I High speed centrifuge rotorBeckman Thermo-mixer Eppendorf 5350-0000-013 Thermo-cycler MJ orEppendorf PCR tubes VWR 53509-304 96 well PCR plate VWR

Required Buffer Recipes are shown below.

-   -   50× TAE buffer        -   242 g Tris base        -   57.1 ml glacial acetic acid        -   37.2 g Na2EDTA-2H₂O        -   Add distilled H₂O to final volume of 1 liter    -   1× TAE buffer        -   20 ml 50× TAE buffer        -   800 ml distilled H₂O    -   0.1 M DTT        -   1.54 g of DTT        -   10 ml of distilled H₂O        -   Store in −20° C.    -   80% Glycerol        -   20 ml Glycerol        -   80 ml distilled H₂O        -   Sterilize by autoclaving    -   4% Agarose Gel with Ethidium Bromide        -   4 g LE agarose        -   100 ml 1× TAE buffer        -   Melt the agarose in a microwave oven and swirl to ensure            even mixing        -   Cool agarose to 55° C.        -   Add 2.5 μl of 20 mg/ml Ethidium Bromide to agarose        -   Pour onto a gel platform

Procedures.

Oligonucleotides were ordered from IDT (1 μmol scale, PAGE purified,lyophilized and 5′ phosphorylated). Lyophilized oligos were spun down inmicrocentrifuge at 12,000×g for 30 seconds before opening the tubes.Resuspend Oligos were resuspended in nuclease-free H₂O at 100 pMole/μlaccording to the data obtained from IDT. Suspended oligos were incubatedat 37° C. for 30 min in a thermomixer at 1,000 RPM. The re-suspendedoligos were spun down in microcentrifuge at 12,000×g for 30 seconds andcombined with 75 μl of matching forward and reverse primers in thin-wallPCR tubes (or 96 well PCR plates). The oligonucleotides were annealed ina thermocycler using the following temperature profile:

-   -   5′ at 94° C.→5′ at 90° C.→5′ at 85° C.→5′ at 80° C.→5′ at 75°        C.→5′ at 70° C.→5′ at 65° C.→5′ at 60° C.→5′ at 55° C.→5′ at 50°        C.→5′ at 45° C.→5′ at 40° C.→5′ at 35° C.→5′ at 30° C.

The final concentration for the annealed DNA fragment concentration was50 pMole/μl. The annealed DNA fragments were stored at −20° C.

Quality control analysis of dsDNA fragments (or fragment pools), wasperformed by setting up the following reactions in 1.5 mlmicro-centrifuge tubes:

dsDNA fragments  1 μl Water 20 μl Sample loading buffer  1 μl Total 22μl

Ten μl of each sample was loaded onto a 4% agarose TAE gel with 0.5μg/ml Ethidium Bromide; a 25-bp DNA ladder was used as a standard. Thegels were run at 100V for 20-30 minutes in 1× TAE buffer.

Standard references for the procedures include Current Protocols inMolecular biology. Edited by Ausubel, F., Brent, R., Kingston, R. E.,Moore, D. D., Seidman, J. G., Smith, J. A., Struhl, K. John Wiley & SonsInc.; Whitehouse, A., Deeble, J., Parmar, R., Taylor, G. R., Markham, A.F., and Meredith, D. M., Analysis of the mismatch and insertion/deletionbinding properties of Thermus thermophilus, HB8, MutS. Bioichemical andBiophysical Research Communications 233, 834-837; Wang, J. and L. J.Directly fishing out subtle mutations in genomic DNA withhistidine-tagged Thermus thermophilus MutS. Mutation Research 547 (2004)41-47.

Example 2. Liquid Phase Synthesis of Combinatorial Variable DomainLibraries-Heavy Chain

This protocol describes the assembly of a humanized heavy chain (HC)variable domain library. The library contains human heavy chainframeworks (FW) and non-human complementarity determining regions (CDR)in the order of: FW1-CDR1-FW2-CDR2-FW3-CDR3. There are total of 7 FW1, 5FW2 and 8 FW3 fragments. The library is assembled by using step wiseliquid phase ligation of FW and CDR DNA fragments. A typical time ofcompletion for this protocol is four days with creation of about fourheavy chain libraries per person.

TABLE 2 Reagents, Consumables, and Equipment Item Approved Catalogue #Description Supplier No. 1 1M Tris/HCl, pH 8.0 - 100 ml Ambion 9855G 20.5M EDTA, pH 8.0 - 100 ml Ambion 9260G 3 5M NaCl - 500 ml Ambion 9759 4Tris base Fisher BP154-1 5 Glacial acetic acid - 500 ml Fisher BP11855006 Na₂EDTA-2H₂O Sigma-Aldrich E9884 7 Nuclease free water - 1000 mlAmbion 9932 8 SeaKem LE agarose Cambrex 50004 9 Ethidium bromideSigma-Aldrich E1510 10 Agarose gel power supply Bio-Rad 1645050 11Agarose gel appratus Bio-Rad 1707764 12 3% and 4% agarose gel SeeAppendix 1 N/A 13 Agarose gel loading buffer Invitrogen 10816-015 14 1kB plus DNA ladder Invitrogen 10787-026 15 Microcentrifuge Eppendorf5417C 16 Thermomixer Eppendorf 5350-0000-013 17 Labquake tube shakersVWR 56264-306 18 Aluminum foil In-house N/A 19 14 ml sterile Falconpoly- VWR 60819-761 propylene round bottom tube 20 1.5 mlmicrocentrifuge tube ISC Bioexpress I5019-07 21 PCR tubes strip withlids VWR 53509-304 22 T4 DNA ligase (20,000 units) NewEngland M0202MBiolab 23 10 mM rATP Promega P1132

Required Buffer Recipes are shown below.

-   -   50× TAE buffer        -   242 g Tris base        -   57.1 ml glacial acetic acid        -   37.2 g Na₂EDTA-2H₂O        -   Add distilled H₂O to final volume of 1 liter    -   1× TAE buffer        -   20 ml 50× TAE buffer        -   800 ml distilled H₂O    -   3% Agarose Gel with ethidium bromide        -   3 g LE agarose        -   100 ml 1× TAE buffer        -   Melt the agarose in a microwave oven and swirl to ensure            even mixing        -   Cool agarose to 55° C.        -   Add 2.5 μl of 20 mg/ml Ethidium Bromide to agarose        -   Pour onto a gel platform    -   4% Agarose Gel with ethidium bromide        -   4 g LE agarose        -   100 ml 1× TAE buffer        -   Melt the agarose in a microwave oven and swirl to ensure            even mixing        -   Cool agarose to 55° C.        -   Add 2.5 μl of 20 mg/ml Ethidium Bromide to agarose        -   Pour onto a gel platform

The liquid phase synthesis procedures were followed as shown in stepwiseformat below. On Day 1, assembly of HC Variable Domain involvesperforming Ligation 1 and Ligation 2 at the same time, and performingLigation 3 and Ligation 4 at the same time.

Ligation 1: FW1b→FW1a

Prepare the following ligation reactions in microcentrifuge tubes onice. There are 7 ligation reactions (FW1-1 to FW1-7). Prepare eachligation reaction in a different microcentrifuge tube, total of 7 tubes.

FW1a fragments (250 pMole) x μL FW1b fragments (250 pMole) x μL 10X T4ligase Buffer 2 μL 10 mM rATP 1 μL Nuclease-free water QS to 19 μL T4ligase 1 μL Total reaction volume 20 μL 

-   1. Mix gently and spin briefly (5 sec.) in microfuge.-   2. Incubate at room temperature for 1 hour.-   3. Set up the following reactions in a 1.5 ml micro-centrifuge tube:

FW1 ligations 20 μl 10x Sample loading buffer  3 μl Total Volume 23 μl

-   4. Load onto a 4% agarose TAE gel with 0.5 μg/ml Ethidium Bromide.    Use 25 bp DNA ladder as standard. Run the gel at 100V for 20-30    minutes in 1× TAE buffer.-   5. Cut out the bands corresponding to the correct sizes and purified    using QIAquick Gel Extraction Kit.-   6. Combine gel fragments from the 7 ligation reactions in two    microcentrifuge tubes.-   7. Add 3 volume of buffer QG to 1 volume of gel.-   8. Incubate at 50° C. for 10 minutes until the gel slice has    completely dissolved. Add 1 gel volume of isopropanol to the sample    and mix.-   9. Place a QIAquick spin column in a provided 2 ml collection tube.-   10. Apply the sample to the QIAquick column, and centrifuge for 1    minute.-   11. Discard flow-through and place QIAquick column back in the same    collection tube.-   12. Add 0.75 ml of buffer PE to QIAquick column and centrifuge for 1    minute.-   13. Discard the flow-through and centrifuge the QIAquick column for    an additional 1 minute at 17,900×g (13,000 rpm).-   14. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.-   15. Add 52 μl of buffer EB to the center of the QIAquick membrane    and centrifuge the column for 1 minute. Let the column stand for 1    minute, and then centrifuge for 1 minute.-   16. Combine the eluted DNA (total volume of 104 μl) and load 6 μl on    4% agarose gel to QC the purified ligation products.

Ligation 2: FW3b→FW3a

-   17. Prepare the following ligation reactions in microcentrifuge    tubes on ice. There are 8 ligation reactions (FW3-1 to FW3-8).    Prepare each ligation reaction in a different microcentrifuge tube,    total of 7 tubes.

FW3a fragments (250 pMole) x μL FW3b fragments (250 pMole) x μL 10X T4ligase Buffer 2 μL 10 mM rATP 1 μL Nuclease-free water QS to 19 μL T4ligase 1 μL Total reaction volume 20 μL 

-   18. Mix gently and spin briefly (5 sec.) in microfuge.-   19. Incubate at room temperature for 1 hour.-   20. Set up the following reactions in a 1.5 ml micro-centrifuge    tube:

FW 3 ligations 20 μL 10x Sample loading buffer  3 μL Total Volume 23 μL

-   21. Load onto a 4% agarose TAE gel with 0.5 μg/ml Ethidium Bromide.    Use 25 bp DNA ladder as standard. Run the gel at 100V for 20-30    minutes in 1× TAE buffer.-   22. Cut out the bands corresponding to the correct sizes and    purified using QIAquick Gel Extraction Kit.-   23. Combine gel fragments from the 7 ligation reactions in two    microcentrifuge tubes.-   24. Add 3 volume of buffer QG to 1 volume of gel.-   25. Incubate at 50° C. for 10 minutes until the gel slice has    completely dissolved. Add 1 gel volume of isopropanol to the sample    and mix.-   26. Place a QIAquick spin column in a provided 2 ml collection tube.-   27. Apply the sample to the QIAquick column, and centrifuge for 1    minute.-   28. Discard flow-through and place QIAquick column back in the same    collection tube.-   29. Add 0.75 ml of buffer PE to QIAquick column and centrifuge for 1    minute.-   30. Discard the flow-through and centrifuge the QIAquick column for    an additional 1 minute at 17,900×g (13,000 rpm).-   31. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.-   32. Add 52 μl of buffer EB to the center of the QIAquick membrane    and centrifuge the column for 1 minute. Let the column stand for 1    minute, and then centrifuge for 1 minutes.-   33. Combine the eluted DNA (total volume of 104 μl) and load 6 μl on    4% agarose gel to QC.

Ligation 3: CDR1→FW1

-   1. Prepare ligation reaction in a microcentrifuge tube on ice:

CDR1 fragments (1 nMole) x μL Gel purified combined FW1 fragments 94 μL 10X T4 ligase Buffer 14 μL  10 mM rATP 1 μL Nuclease-free water QS to139 μL T4 ligase 1 μL Total reaction volume 140 μL 

-   2. Mix gently and spin briefly (5 sec.) in microfuge.-   3. Incubate at room temperature for 1 hour.-   4. Set up the following reactions in a 1.5 ml micro-centrifuge tube:

CDR1-FW 1 ligations 140 μl 10x Sample loading buffer  15 μl Total Volume155 μl

-   5. Load onto a 4% agarose TAE gel with 0.5 μg/ml Ethidium Bromide.    Use 25 bp DNA ladder as standard. Run the gel at 100V for 20-30    minutes in 1× TAE buffer.-   6. Cut out the bands corresponding to the correct sizes and purified    using the QIAquick Gel Extraction Kit.-   7. Combine the gel fragments in two microcentrifuge tubes.-   8. Add 3 volume of buffer QG to 1 volume of gel.-   9. Incubate at 50° C. for 10 minutes until the gel slice has    completely dissolved. Add 1 gel volume of isopropanol to the sample    and mix.-   10. Place a QIAquick spin column in a provided 2 ml collection tube.-   11. Apply the sample to the QIAquick column, and centrifuge for 1    minute.-   12. Discard flow-through and place QIAquick column back in the same    collection tube.-   13. Add 0.75 ml of buffer PE to QIAquick column and centrifuge for 1    minute.-   14. Discard the flow-through and centrifuge the QIAquick column for    an additional 1 minute at 17,900×g (13,000 rpm).-   15. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.-   16. Add 52 μl of buffer EB to the center of the QIAquick membrane    and centrifuge the column for 1 minute. Let the column stand for 1    minute, and then centrifuge for 1 minute.-   17. Combine the eluted DNA (total volume of 104 μl) and load 6 ul on    4% agarose gel to QC. Ligation 4: CDR2→FW3-   18. Prepare ligation reaction in a microcentrifuge tube on ice:

CDR2 fragments (1 nMole) x μL Gel purified combined FW3 fragments 94 μL 10X T4 ligase Buffer 14 μL  10 mM rATP 1 μL Nuclease-free water QS to139 μL T4 ligase 1 μL Total reaction volume 140 μL 

-   19. Mix gently and spin briefly (5 sec.) in microfuge.-   20. Incubate at room temperature for 1 hour.-   21. Set up the following reactions in a 1.5 ml micro-centrifuge    tube:

CDR2-FW3 ligations 140 μl 10x Sample loading buffer  15 μl Total Volume155 μl

-   22. Load onto a 4% agarose TAE gel with 0.5 μg/ml Ethidium Bromide.    Use 25 bp DNA ladder as standard. Run the gel at 100V for 20-30    minutes in 1× TAE buffer.-   23. Cut out the bands corresponding to the correct sizes and    purified using the QIAquick Gel Extraction Kit.-   24. Combine the gel fragments in two microcentrifuge tubes.-   25. Add 3 volume of buffer QG to 1 volume of gel.-   26. Incubate at 50° C. for 10 minutes until the gel slice has    completely dissolved. Add 1 gel volume of isopropanol to the sample    and mix.-   27. Place a QIAquick spin column in a provided 2 ml collection tube.-   28. Apply the sample to the QIAquick column, and centrifuge for 1    minute.-   29. Discard flow-through and place QIAquick column back in the same    collection tube.-   30. Add 0.75 ml of buffer PE to QIAquick column and centrifuge for 1    minute.-   31. Discard the flow-through and centrifuge the QIAquick column for    an additional 1 minute at 17,900×g (13,000 rpm).-   32. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.-   33. Add 52 μl of buffer EB to the center of the QIAquick membrane    and centrifuge the column for 1 minute. Let the column stand for 1    minute, and then centrifuge for 1 minute.-   34. Combine the eluted DNA (total volume of 104 μl) and load 6 μl on    4% agarose gel to QC.

On Day 2, assembly of HC variable domain was continued by performingLigation 5 and Ligation 6 at the same time

Ligation 5: FW2→CDR1-FW1

-   1. Prepare ligation reaction in a microcentrifuge tube on ice:

FW2 fragment pool (450 pMole) x μL Gel purified CDR1-FW1 fragments 94μL  10X T4 ligase Buffer 14 μL  10 mM rATP 1 μL Nuclease-free water QSto 139 μL T4 ligase 1 μL Total reaction volume 140 μL The FW2 fragment pool contained 5 FW2 fragments, each at 90 pMole.

-   2. Mix gently and spin briefly (5 sec.) in microfuge.-   3. Incubate at room temperature for 1 hour.-   4. Set up the following reactions in a 1.5 ml micro-centrifuge tube:

FW2-CDR-1-FW1 ligations 140 μl 10x Sample loading buffer  15 μl TotalVolume 155 μl

-   5. Load onto a 4% agarose TAE gel with 0.5 μg/ml Ethidium Bromide.    Use 25 bp DNA ladder as standard. Run the gel at 100V for 20-30    minutes in 1× TAE buffer.-   6. Cut out the bands corresponding to the correct sizes and purified    using QIAquick Gel Extraction Kit.-   7. Combine gel fragments from the 7 ligation reactions in two    microcentrifuge tubes.-   8. Add 3 volume of buffer QG to 1 volume of gel.-   9. Incubate at 50° C. for 10 minutes until the gel slice has    completely dissolved. Add 1 gel volume of isopropanol to the sample    and mix.-   10. Place a QIAquick spin column in a provided 2 ml collection tube.-   11. Apply the sample to the QIAquick column, and centrifuge for 1    minute.-   12. Discard flow-through and place QIAquick column back in the same    collection tube.-   13. Add 0.75 ml of buffer PE to QIAquick column and centrifuge for 1    minute.-   14. Discard the flow-through and centrifuge the QIAquick column for    an additional 1 minute at 17,900×g (13,000 rpm).-   15. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.-   16. Add 30 μL of buffer EB to the center of the QIAquick membrane    and centrifuge the column for 1 minute. Let the column stand for 1    minute, and then centrifuge for 1 minutes.-   17. Combine the eluted DNA (total volume of 60 μL) and load 3 μL on    4% agarose gel to QC.

Ligation 6: CDR3→FW3-CDR2

-   18. Prepare ligation reaction in a microcentrifuge tube on ice:

CDR3 fragment pool (500 pMole) x μL Gel purified FW3-CDR2 fragments 94μL  10X T4 ligase Buffer 14 μL  10 mM rATP 1 μL Nuclease-free water QSto 139 μL T4 ligase 1 μL Total reaction volume 140 μL [[The FW2 fragment pool contained 5 FW2 fragments, each at 90 pMole]]

-   19. Mix gently and spin briefly (5 sec.) in microfuge.-   20. Incubate at room temperature for 1 hour.-   21. Set up the following reactions in a 1.5 ml micro-centrifuge    tube:

CDR3-FW3-CDR2 ligations 140 μl 10x Sample loading buffer  15 μl TotalVolume 155 μl

-   22. Load onto a 4% agarose TAE gel with 0.5 μg/ml Ethidium Bromide.    Use 25 bp DNA ladder as standard. Run the gel at 100V for 20-30    minutes in 1× TAE buffer.-   23. Cut out the bands corresponding to the correct sizes and    purified using QIAquick Gel Extraction Kit.-   24. Combine gel fragments from the 7 ligation reactions in two    microcentrifuge tubes.-   25. Add 3 volume of buffer QG to 1 volume of gel.-   26. Incubate at 50° C. for 10 minutes until the gel slice has    completely dissolved. Add 1 gel volume of isopropanol to the sample    and mix.-   27. Place a QIAquick spin column in a provided 2 ml collection tube.-   28. Apply the sample to the QIAquick column, and centrifuge for 1    minute.-   29. Discard flow-through and place QIAquick column back in the same    collection tube.-   30. Add 0.75 ml of buffer PE to QIAquick column and centrifuge for 1    minute.-   31. Discard the flow-through and centrifuge the QIAquick column for    an additional 1 minute at 17,900×g (13,000 rpm).-   32. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.-   33. Add 30 μL of buffer EB to the center of the QIAquick membrane    and centrifuge the column for 1 minute. Let the column stand for 1    minute, and then centrifuge for 1 minute.-   34. Combine the eluted DNA (total volume of 60 μL) and load 3 μL on    4% agarose gel to QC.

Ligation 7: Full Length HC Variable Domain

-   1. Prepare ligation reactions in a microcentrifuge tube on ice:

FW1-CDR1-FW2 fragments 49 μL CDR2-FW3-CDR3 fragments 49 μL 10X T4 ligaseBuffer 12 μL 10 mM rATP  5 μL Nuclease-free water QS to 345 μL T4 ligase 5 μL Total reaction volume 350 μL 

-   35. Mix gently and spin briefly (5 sec.) in microfuge.-   36. Incubate at room temperature for 1 hour.-   37. Set up the following reactions in a 1.5 ml micro-centrifuge    tube:

Full length HC variable domain ligations 140 μl 10x Sample loadingbuffer  15 μl Total Volume 155 μl

-   38. Load onto a 3% agarose TAE gel with 0.5 μg/ml Ethidium Bromide.    Use 100 bp DNA ladder as standard. Run the gel at 100V for 20-30    minutes in 1× TAE buffer.-   39. Cut out the bands corresponding to the correct sizes and    purified using QIAquick Gel Extraction Kit.-   40. Combine gel fragments in one microcentrifuge tube.-   41. Add 3 volume of buffer QG to 1 volume of gel.-   42. Incubate at 50° C. for 10 minutes until the gel slice has    completely dissolved. Add 1 gel volume of isopropanol to the sample    and mix.-   43. Place a QIAquick spin column in a provided 2 ml collection tube.-   44. Apply the sample to the QIAquick column, and centrifuge for 1    minute.-   45. Discard flow-through and place QIAquick column back in the same    collection tube.-   46. Add 0.75 ml of buffer PE to QIAquick column and centrifuge for 1    minute.-   47. Discard the flow-through and centrifuge the QIAquick column for    an additional 1 minute at 17,900×g (13,000 rpm).-   48. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.-   49. Add 30 μl of buffer EB to the center of the QIAquick membrane    and centrifuge the column for 1 minute. Let the column stand for 1    minute, and then centrifuge for 1 minute.-   50. Load 3 μl on 3% agarose gel to QC.

Determining the heavy chain CDRs. The following set of rules allows theidentification of the CDRs in most antibody heavy chain variable domainsequences.

CDR-H1

Start: — position 26, always 4 after a cysteine residueResidues before: C-X-X-XLength: 10-12 amino acidsResidues after: always a W, usually W-V, but also W-I, W-A

CDR-H2

Start: always 15 residues after the end of CDR-H1Residues before: usually L-E-W-I-G, but a number of variationsLength: 16-19 amino acidsResidues after: K/R-L/I/V/F/T/A-T/S/I/A

CDR-H3

Start: always 33 residues after end of CDR-H2 (always 2 after acysteine)Residues before: always C-X-X, usually C-A-RLength: 3-25 residuesResidues after: W-G-X-G (typically W-G-Q-G)

References for this protocol include L. B. K. C. Antibody humanizationby CDR grafting. Antibody Engineering, Methods and protocols. Edit byBenny K. C. Lo, Methods in Molecular Biology, 2004; 248, 135-159;Developing a minimally immunogenic humanized antibody by SDR grafting.Antibody Engineering, Methods and protocols. Edit by Benny K. C. Lo,Methods in Molecular Biology, 2004, 248, 361-376; Bassette, P. H., Mena,M. A., Nguyen, A. W. and Daugherty, P. S. Construction of DesignedProtein Libraries Using Gene Assembly Mutagenesis. Directed EvolutionLibrary Creation, Methods and protocols. Edit by Frances H. Arnold andGeorge Georgiou, Methods in Molecular Biology, 2003, 231, 29-37; Chames,P., Hoogenboom, H. R., and Henderikx, P. Selection on Biotinylatedantigens. Antibody Engineering, Edit by R. Kontermann and S. Dubel,Springer Lab Manual, 149-166; Obrien S., and Jones, T. Humanisingantibodies by CDR grafting. Antibody Engineering, Edit by R. Kontermannand S. Dubel, Springer Lab Manual, 567-590.

Example 3. Liquid Phase Synthesis of Combinatorial Variable DomainLibraries—Light Chain

This protocol describes the assembly of a humanized light chain (LC)variable domain library. The library contains human light chainframeworks (FW) and non-human complementarity determining regions (CDR)in the order of: FW1-CDR1-FW2-CDR2-FW3-CDR3. There are total of 7 FW1, 4FW2 and 8 FW3 fragments. The library is assembled by using step wiseliquid phase ligation of FW and CDR DNA fragments. A typical time ofcompletion for this protocol is four days with creation of about fourheavy chain libraries per person. Reagents, consumables and equipment isdescribed in Example 2, Table 2, above. Required Buffer Recipes areshown above in Example 2.

The liquid phase synthesis procedures were followed as shown in stepwiseformat below. On Day 1, assembly of HC Variable Domain involvesperforming Ligation 1 and Ligation 2 at the same time, and performingLigation 3 and Ligation 4 at the same time.

Ligation 1: FW1b→FW1a

-   34. Prepare the following ligation reactions in microcentrifuge    tubes on ice. There are 7 ligation reactions (FW1-1 to FW1-7).    Prepare each ligation reaction in a different microcentrifuge tube,    total of 7 tubes.

FW1a fragments (250 pMole) x μL FW1b fragments (250 pMole) x μL 10X T4ligase Buffer 2 μL 10 mM rATP 1 μL Nuclease-free water QS to 19 μL T4ligase 1 μL Total reaction volume 20 μL 

-   35. Mix gently and spin briefly (5 sec.) in microfuge.-   36. Incubate at room temperature for 1 hour.-   37. Set up the following reactions in a 1.5 ml micro-centrifuge    tube:

FW1 ligations 20 μl 10x Sample loading buffer  3 μl Total Volume 23 μl

-   38. Load onto a 4% agarose TAE gel with 0.5 μg/ml Ethidium Bromide.    Use 25 bp DNA ladder as standard. Run the gel at 100V for 20-30    minutes in 1× TAE buffer.-   39. Cut out the bands corresponding to the correct sizes and    purified using QIAquick Gel Extraction Kit.-   40. Combine gel fragments from the 7 ligation reactions in two    microcentrifuge tubes.-   41. Add 3 volume of buffer QG to 1 volume of gel.-   42. Incubate at 50° C. for 10 minutes until the gel slice has    completely dissolved. Add 1 gel volume of isopropanol to the sample    and mix.-   43. Place a QIAquick spin column in a provided 2 ml collection tube.-   44. Apply the sample to the QIAquick column, and centrifuge for 1    minute.-   45. Discard flow-through and place QIAquick column back in the same    collection tube.-   46. Add 0.75 ml of buffer PE to QIAquick column and centrifuge for 1    minute.-   47. Discard the flow-through and centrifuge the QIAquick column for    an additional 1 minute at 17,900×g (13,000 rpm).-   48. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.-   49. Add 52 μl of buffer EB to the center of the QIAquick membrane    and centrifuge the column for 1 minute. Let the column stand for 1    minute, and then centrifuge for 1 minute.-   50. Combine the eluted DNA (total volume of 104 μl) and load 6 μl on    4% agarose gel to QC the purified ligation products.

Ligation 2: FW3b→FW3a

-   51. Prepare the following ligation reactions in microcentrifuge    tubes on ice. There are 8 ligation reactions (FW3-1 to FW3-8).    Prepare each ligation reaction in a different microcentrifuge tube,    total of 7 tubes.

FW3a fragments (250 pMole) x μL FW3b fragments (250 pMole) x μL 10X T4ligase Buffer 2 μL 10 mM rATP 1 μL Nuclease-free water QS to 19 μL T4ligase 1 μL Total reaction volume 20 μL 

-   52. Mix gently and spin briefly (5 sec.) in microfuge.-   53. Incubate at room temperature for 1 hour.-   54. Set up the following reactions in a 1.5 ml micro-centrifuge    tube:

FW 3 ligations 20 μL 10x Sample loading buffer  3 μL Total Volume l 23μL

-   55. Load onto a 4% agarose TAE gel with 0.5 μg/ml Ethidium Bromide.    Use 25 bp DNA ladder as standard. Run the gel at 100V for 20-30    minutes in 1× TAE buffer.-   56. Cut out the bands corresponding to the correct sizes and    purified using QIAquick Gel Extraction Kit.-   57. Combine gel fragments from the 7 ligation reactions in two    microcentrifuge tubes.-   58. Add 3 volume of buffer QG to 1 volume of gel.-   59. Incubate at 50° C. for 10 minutes until the gel slice has    completely dissolved. Add 1 gel volume of isopropanol to the sample    and mix.-   60. Place a QIAquick spin column in a provided 2 ml collection tube.-   61. Apply the sample to the QIAquick column, and centrifuge for 1    minute.-   62. Discard flow-through and place QIAquick column back in the same    collection tube.-   63. Add 0.75 ml of buffer PE to QIAquick column and centrifuge for 1    minute.-   64. Discard the flow-through and centrifuge the QIAquick column for    an additional 1 minute at 17,900×g (13,000 rpm).-   65. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.-   66. Add 52 μL of buffer EB to the center of the QIAquick membrane    and centrifuge the column for 1 minute. Let the column stand for 1    minute, and then centrifuge for 1 minutes.-   67. Combine the eluted DNA (total volume of 104 μL) and load 6 uLon    4% agarose gel to QC.

Ligation 3: CDR1→FW1

-   35. Prepare ligation reaction in a microcentrifuge tube on ice:

CDR1 fragments (1 nMole) x μL Gel purified combined FW1 fragments 94 μL 10X T4 ligase Buffer 14 μL  10 mM rATP 1 μL Nuclease-free water QS to139 μL T4 ligase 1 μL Total reaction volume 140 μL 

-   36. Mix gently and spin briefly (5 sec.) in microfuge.-   37. Incubate at room temperature for 1 hour.-   38. Set up the following reactions in a 1.5 ml micro-centrifuge    tube:

CDR1-FW 1 ligations 140 μl 10x Sample loading buffer  15 μl Total Volume155 μl

-   39. Load onto a 4% agarose TAE gel with 0.5 μg/ml Ethidium Bromide.    Use 25 bp DNA ladder as standard. Run the gel at 100V for 20-30    minutes in 1× TAE buffer.-   40. Cut out the bands corresponding to the correct sizes and    purified using the QIAquick Gel Extraction Kit.-   41. Combine the gel fragments in two microcentrifuge tubes.-   42. Add 3 volume of buffer QG to 1 volume of gel.-   43. Incubate at 50° C. for 10 minutes until the gel slice has    completely dissolved. Add 1 gel volume of isopropanol to the sample    and mix.-   44. Place a QIAquick spin column in a provided 2 ml collection tube.-   45. Apply the sample to the QIAquick column, and centrifuge for 1    minute.-   46. Discard flow-through and place QIAquick column back in the same    collection tube.-   47. Add 0.75 ml of buffer PE to QIAquick column and centrifuge for 1    minute.-   48. Discard the flow-through and centrifuge the QIAquick column for    an additional 1 minute at 17,900×g (13,000 rpm).-   49. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.-   50. Add 52 □l of buffer EB to the center of the QIAquick membrane    and centrifuge the column for 1 minute. Let the column stand for 1    minute, and then centrifuge for 1 minute.-   51. Combine the eluted DNA (total volume of 104 □l) and load 6 ul on    4% agarose gel to QC.

Ligation 4: CDR2→FW3

-   52. Prepare ligation reaction in a microcentrifuge tube on ice:

CDR2 fragments (1 nMole) x μL Gel purified combined FW3 fragments 94 μL 10X T4 ligase Buffer 14 μL  10 mM rATP 1 μL Nuclease-free water QS to139 μL T4 ligase 1 μL Total reaction volume 140 μL 

-   53. Mix gently and spin briefly (5 sec.) in microfuge.-   54. Incubate at room temperature for 1 hour.-   55. Set up the following reactions in a 1.5 ml micro-centrifuge    tube:

CDR2-FW3 ligations 140 μl 10x Sample loading buffer  15 μl Total Volume155 μl

-   56. Load onto a 4% agarose TAE gel with 0.5 μg/ml Ethidium Bromide.    Use 25 bp DNA ladder as standard. Run the gel at 100V for 20-30    minutes in 1× TAE buffer.-   57. Cut out the bands corresponding to the correct sizes and    purified using the QIAquick Gel Extraction Kit.-   58. Combine the gel fragments in two microcentrifuge tubes.-   59. Add 3 volume of buffer QG to 1 volume of gel.-   60. Incubate at 50° C. for 10 minutes until the gel slice has    completely dissolved. Add 1 gel volume of isopropanol to the sample    and mix.-   61. Place a QIAquick spin column in a provided 2 ml collection tube.-   62. Apply the sample to the QIAquick column, and centrifuge for 1    minute.-   63. Discard flow-through and place QIAquick column back in the same    collection tube.-   64. Add 0.75 ml of buffer PE to QIAquick column and centrifuge for 1    minute.-   65. Discard the flow-through and centrifuge the QIAquick column for    an additional 1 minute at 17,900×g (13,000 rpm).-   66. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.-   67. Add 52 μL of buffer EB to the center of the QIAquick membrane    and centrifuge the column for 1 minute. Let the column stand for 1    minute, and then centrifuge for 1 minute.-   68. Combine the eluted DNA (total volume of 104 μL) and load 6 uLon    4% agarose gel to QC.

On Day 2, assembly of HC variable domain was continued by performingLigation 5 and Ligation 6 at the same time

Ligation 5: FW2→CDR1-FW1

-   51. Prepare ligation reaction in a microcentrifuge tube on ice:

FW2 fragment pool (450 pMole) x μL Gel purified CDR1-FW1 fragments 94μL  10X T4 ligase Buffer 14 μL  10 mM rATP 1 μL Nuclease-free water QSto 139 μL T4 ligase 1 μL Total reaction volume 140 μL The FW2 fragment pool contained 5 FW2 fragments, each at 90 pMole.

-   52. Mix gently and spin briefly (5 sec.) in microfuge.-   53. Incubate at room temperature for 1 hour.-   54. Set up the following reactions in a 1.5 ml micro-centrifuge    tube:

FW2-CDR-1-FW1 ligations 140 μl 10x Sample loading buffer  15 μl TotalVolume 155 μl

-   55. Load onto a 4% agarose TAE gel with 0.5 μg/ml Ethidium Bromide.    Use 25 bp DNA ladder as standard. Run the gel at 100V for 20-30    minutes in 1× TAE buffer.-   56. Cut out the bands corresponding to the correct sizes and    purified using QIAquick Gel Extraction Kit.-   57. Combine gel fragments from the 7 ligation reactions in two    microcentrifuge tubes.-   58. Add 3 volume of buffer QG to 1 volume of gel.-   59. Incubate at 50° C. for 10 minutes until the gel slice has    completely dissolved. Add 1 gel volume of isopropanol to the sample    and mix.-   60. Place a QIAquick spin column in a provided 2 ml collection tube.-   61. Apply the sample to the QIAquick column, and centrifuge for 1    minute.-   62. Discard flow-through and place QIAquick column back in the same    collection tube.-   63. Add 0.75 ml of buffer PE to QIAquick column and centrifuge for 1    minute.-   64. Discard the flow-through and centrifuge the QIAquick column for    an additional 1 minute at 17,900×g (13,000 rpm).-   65. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.-   66. Add 30 μL of buffer EB to the center of the QIAquick membrane    and centrifuge the column for 1 minute. Let the column stand for 1    minute, and then centrifuge for 1 minutes.-   67. Combine the eluted DNA (total volume of 60 μL) and load 3 μL on    4% agarose gel to QC.

Ligation 6: CDR3→FW3-CDR2

-   68. Prepare ligation reaction in a microcentrifuge tube on ice:

CDR3 fragment pool (500 pMole) x μL Gel purified FW3-CDR2 fragments 94μL  10X T4 ligase Buffer 14 μL  10 mM rATP 1 μL Nuclease-free water QSto 139 μL T4 ligase 1 μL Total reaction volume 140 μL [[The FW2 fragment pool contained 4 FW2 fragments, each at 90 pMole]]

-   69. Mix gently and spin briefly (5 sec.) in microfuge.-   70. Incubate at room temperature for 1 hour.-   71. Set up the following reactions in a 1.5 ml micro-centrifuge    tube:

CDR3-FW3-CDR2 ligations 140 μl 10x Sample loading buffer  15 μl TotalVolume 155 μl

-   72. Load onto a 4% agarose TAE gel with 0.5 μg/ml Ethidium Bromide.    Use 25 bp DNA ladder as standard. Run the gel at 100V for 20-30    minutes in 1× TAE buffer.-   73. Cut out the bands corresponding to the correct sizes and    purified using QIAquick Gel Extraction Kit.-   74. Combine gel fragments from the 7 ligation reactions in two    microcentrifuge tubes.-   75. Add 3 volume of buffer QG to 1 volume of gel.-   76. Incubate at 50° C. for 10 minutes until the gel slice has    completely dissolved. Add 1 gel volume of isopropanol to the sample    and mix.-   77. Place a QIAquick spin column in a provided 2 ml collection tube.-   78. Apply the sample to the QIAquick column, and centrifuge for 1    minute.-   79. Discard flow-through and place QIAquick column back in the same    collection tube.-   80. Add 0.75 ml of buffer PE to QIAquick column and centrifuge for 1    minute.-   81. Discard the flow-through and centrifuge the QIAquick column for    an additional 1 minute at 17,900×g (13,000 rpm).-   82. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.-   83. Add 30 μL of buffer EB to the center of the QIAquick membrane    and centrifuge the column for 1 minute. Let the column stand for 1    minute, and then centrifuge for 1 minute.-   84. Combine the eluted DNA (total volume of 60 μL) and load 3 μL on    4% agarose gel to QC.

Ligation 7: Full Length LC Variable Domain

-   2. Prepare ligation reactions in a microcentrifuge tube on ice:

FW1-CDR1-FW2 fragments 49 μL CDR2-FW3-CDR3 fragments 49 μL 10X T4 ligaseBuffer 12 μL 10 mM rATP  5 μL Nuclease-free water QS to 345 μL T4 ligase 5 μL Total reaction volume 350 μL 

-   85. Mix gently and spin briefly (5 sec.) in microfuge.-   86. Incubate at room temperature for 1 hour.-   87. Set up the following reactions in a 1.5 ml micro-centrifuge    tube:

Full length LC variable domain ligations 140 μl 10x Sample loadingbuffer  15 μl Total Volume 155 μl

-   88. Load onto a 3% agarose TAE gel with 0.5 μg/ml Ethidium Bromide.    Use 100 bp DNA ladder as standard. Run the gel at 100V for 20-30    minutes in 1× TAE buffer.-   89. Cut out the bands corresponding to the correct sizes and    purified using QIAquick Gel Extraction Kit.-   90. Combine gel fragments in one microcentrifuge tube.-   91. Add 3 volume of buffer QG to 1 volume of gel.-   92. Incubate at 50° C. for 10 minutes until the gel slice has    completely dissolved. Add 1 gel volume of isopropanol to the sample    and mix.-   93. Place a QIAquick spin column in a provided 2 ml collection tube.-   94. Apply the sample to the QIAquick column, and centrifuge for 1    minute.-   95. Discard flow-through and place QIAquick column back in the same    collection tube.-   96. Add 0.75 ml of buffer PE to QIAquick column and centrifuge for 1    minute.-   97. Discard the flow-through and centrifuge the QIAquick column for    an additional 1 minute at 17,900×g (13,000 rpm).-   98. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.-   99. Add 30 μl of buffer EB to the center of the QIAquick membrane    and centrifuge the column for 1 minute. Let the column stand for 1    minute, and then centrifuge for 1 minute.-   100. Load 3 μl on 3% agarose gel to QC.

Determining the light chain CDRs. The following set of rules allows theidentification of the CDRs in most antibody light chain variable domainsequences.

CDR-L1

Start: ˜ position 24, always 1 after a cysteine residueResidues before: CLength: 10-17 amino acidsResidues after: always a W, usually W-Y-Q, but also W-L-Q, W-F-Q, W-Y-L

CDR-L2

Start: always 16 residues after the end of CDR-L1Residues before: usually I-Y, but also V-Y, I-K, I-FLength: always 7 amino acids

CDR-L3

Start: always 33 residues after end of CDR-L2 (always 2 after acysteine)Residues before: always CLength: 7-11 residuesResidues after: F-G-X-G (typically F-G-Q-G)

Example 4. Cloning of Assembled Variable Domains. Ligation of HC and LCVariable Domain with Fc

This protocol describes the cloning of assembled humanized heavy andlight chain variable domains into BioAtla expression vectors, pBA-K andpBA-L. The estimated time of completion 7 days with 4 cloning proceduresper person. Table 3 shows required reagents, consumables and equipment.

TABLE 3 Reagents, Consumables, and Equipment Item Approved Catalogue #Description Supplier No. 1 1 kB plus DNA ladder Invitrogen 10787-026 21% agarose gel See Appendix N/A 1 3 1.5 ml microcentrifuge tube ISCI5019-07 Bioexpress 4 14 ml sterile Falcon VWR 60819-761 polypropyleneround bottom tube 5 5M NaCl - 500 ml Ambion 9759 6 Agar 7 Agarose gelappratus Bio-Rad 1707764 8 Agarose gel loading buffer Invitrogen10816-015 9 Agarose gel power supply Bio-Rad 1645050 10 BsaI New EnglandR0535S Biolab 11 BsmBI New England R0580L Biolab 12 Ethidium bromideSigma-Aldrich E1510 13 Glacial acetic acid - 500 ml Fisher BP1185500 14Microcentrifuge Eppendorf 5417C 15 Na₂EDTA-2H₂O Sigma-Aldrich E9884 16Nuclease free water - 1000 ml Ambion 9932 17 PCR tubes strip with lidsVWR 53509-304 18 Plate scraper 19 Plating beads 20 QIAprep Spingminiprep kit Qiagen 27104 21 QIAquick PCR purification Qiagen 28104 kit22 SeaKem LE agarose Cambrex 50004 23 T4 DNA ligase (20,000 units)NewEngland M0202T Biolab 24 Tris base Fisher BP154-1 25 Tryptone 26Water bath 27 XLI Blue Supercompetent cells Stratagene 200236 28 YeastExtract

The following buffers are required for this protocol.

50× TAE buffer

-   -   242 g Tris base    -   57.1 ml glacial acetic acid    -   37.2 g Na₂EDTA-2H₂O    -   Add distilled H₂O to final volume of 1 liter        1× TAE buffer    -   20 ml 50× TAE buffer    -   800 ml distilled H₂O        Agarose Gel with ethidium bromide    -   1 g LE agarose    -   100 ml 1× TAE buffer    -   Melt the agarose in a microwave oven and swirl to ensure even        mixing    -   Cool agarose to 55° C.    -   Add 2.5 μl of 20 mg/ml Ethidium Bromide to agarose    -   Pour onto a gel platform

LB

-   -   10 g NaCl    -   10 g tryptone    -   5 g yeast extract    -   Add distilled H₂O to a final volume of 1 liter    -   Adjust pH to 7.0 with 5 N NaOH    -   Autoclave        LB-carbenicillin agar    -   10 g NaCl    -   10 g tryptone    -   5 g yeast extract    -   20 g agar    -   Add distilled H₂O to a final volume of 1 liter    -   Adjust pH to 7.0 with 5 N NaOH    -   Autoclave    -   Cool to 55° C.    -   Add 10 ml of 10 mg/ml of filter-sterilized carbenicillin    -   Pour into petri dishes (25 ml/100-mm plate)

SOC Medium

-   -   0.5 g NaCl    -   20 g tryptone    -   0.5 g yeast extract    -   2 ml of filter-sterilized 20% glucose    -   Add distilled H₂O to a final volume of 1 liter    -   Autoclave    -   Add 10 ml of filter-sterilized 1 M MgCl₂ and 10 ml of        filter-sterilized 1 M MgSO₈ prior to use

On Day 1 digest pBA vector with BsaI. Prepare the following digestionreaction in a microcentrifuge tube on ice:

pBA (5 ug) x μL 10X NEB Buffer 3 10 μL Nuclease-free water QS to 97 μLBsaI (10 U/μL) 3 μL Total reaction volume 100 μL

-   1. Mix gently and spin briefly (5 sec.) in microfuge-   2. Incubate the reaction at 50° C. overnight

Day 2

-   3. Add 2 μL of Apex phosphatase to the microcentrifuge tube-   4. Incubate at 37° C. for 10 minutes-   5. Heat at 70° C. for 5 minutes to inactivate the Apex phosphatase

Purify BsaI digested pBA vector with QIAquick PCR Purification Kit

-   6. Add 500 μL of Buffer PBI to the microcentrifuge-   7. Mix by vortexing and quick centrifuge-   8. Load 750 μL at a time onto a column-   9. Centrifuge at 12,000×g for 1 minute and decant liquid from    collection tube-   10. Repeat until all sample has been processed.-   11. Wash with 750 μL PE Buffer (Ethanol added!)-   12. Centrifuge at 12,000×g for 1 minute and decant liquid from    collection tube-   13. Place column back onto collection tube and centrifuge again-   14. Put column onto new microcentrifuge tubes and elute with 50 μL    EB Buffer

Quality Control Analysis

-   1. To QC the BsaI digested pBA vector, set up the following    reactions in a 1.5 ml micro-centrifuge tube:

pBA-BsaI 2 μl pBA-uncut 2 μl Water 7 μl Water 7 μl Sample loading buffer1 μl Sample loading buffer 1 μl Total Volume 10 μl  Total Volume 10 μl 

-   2. Load 10 μL onto a 1% agarose TAE gel with 0.5 μg/ml Ethidium    Bromide. Use 1 kb plus DNA ladder as standard. Run the gel at 100V    for 20-30 minutes in 1× TAE buffer.-   3. Determine the concentration of the BsaI digested pBA vector with    spectrophotometer (OD_(260/280))-   4. Set up the following ligation reactions to determine the    background and ligation efficiency of the BsaI digested pBA vector:

a. Vector Background Ligation

pBA-BsaI (100 ng) x μL 5X T4 ligase Buffer 4 μL Nuclease-free water QSto 19 μL T4 ligase (2,000 U/μL) 1 μL Total reaction volume 20 μL

b. Test Insert Ligations

pBA-BsaI (100 ng) x μL Test insert (5 ng, 10 ng) y μL 5X T4 ligaseBuffer 4 μL Nuclease-free water QS to 19 μL T4 ligase (2,000 U/μL) 1 μLTotal reaction volume 20 μL

-   5. Mix gently and spin briefly (5 sec.) in microfuge-   6. Incubate at room temperature for 2 hours or 16° C. overnight-   7. Transform each of the ligation reaction mixtures into XLI Blue    Supercompetent cells-   8. Pre-chill 14 ml BD Falcon polypropylene round-bottom tubes on    ice. Warm SOC medium to 42° C.-   9. Thaw the XLI Blue Supercompetent cells on ice. When thawed,    gently mix and aliquot 100 ul of cells into each of the pre-chilled    tubes.-   10. Add 1.7 μL of beta-mercaptoethanol to each aliquot of cells.    Incubate the cells on ice for 10 minutes, swirling gently every 2    minutes.-   11. Add 2 μL of the ligation reaction mixture to one aliquot of    cells. Flick the tubes gently.-   12. Incubate the tubes on ice for 30 minutes.-   13. Heat-pulse the tubes in a 42° C. water bath for 45 seconds.-   14. Incubate the tubes on ice for 2 minutes-   15. Add 900 ul of preheated SOC medium and incubate the tubes at    37° C. for 1 hour with shaking at 225-250 rpm.-   16. Plate 20 μLand 200 μL of the transformation mixture on LB agar    plates containing carbenicillin.-   17. Incubate the plates at 37° C. overnight.

Day 3

-   18. Count colonies and calculate the efficiency and background of    the vector as well as the optimal amount of insert for ligation.

Ligate Heavy Chain (HC) Variable Domain into BsaI digested pBA vectorPrepare the following ligation reaction in a microcentrifuge tube onice:

pBA-BsaI (100 ng) x μL Heavy chain (HC) variable domain y μL 5X T4ligase Buffer 4 μL Nuclease-free water QS to 19 μL T4 ligase (2,000U/μL) 1 μL Total reaction volume 20 μL

-   1. Mix gently and spin briefly (5 sec.) in microfuge-   2. Incubate at room temperature for 2 hours or 16° C. overnight-   3. Transform each of the ligation reaction mixtures into XLI Blue    Supercompetent cells-   4. Pre-chill 14 ml BD Falcon polypropylene round-bottom tubes on    ice. Prepare SOC medium to 42° C.-   5. Thaw the XLI Blue Supercompetent cells on ice. When thawed,    gently mix and aliquot 100 ul of cells into each of the pre-chilled    tubes.-   6. Add 1.7 μL of beta-mercaptoethanol to each aliquot of cells.    Incubate the cells on ice for 10 minutes, swirling gently every 2    minutes.-   7. Add 2 μL of the ligation reaction mixture to one aliquot of    cells. Flick the tubes gently.-   8. Incubate the tubes on ice for 30 minutes.-   9. Heat-pulse the tubes in a 42° C. water bath for 45 seconds.-   10. Incubate the tubes on ice for 2 minutes-   11. Add 900 ul of preheated SOC medium and incubate the tubes at    37° C. for 1 hour with shaking at 225-250 rpm.-   12. Plate 20 μL and 200 μL of the transformation mixture on LB agar    plates containing carbenicillin.-   13. Incubate the plates at 37° C. overnight.

Day 4

-   14. Count colonies on plates and pick 24 colonies for miniprep and    sequencing.-   15. Repeat transformations to obtain at least 2000 colonies.-   16. Add 6 ml LB medium to each plate containing colonies and gently    scrape the bacteria with a spreader to form a dense suspension.-   17. Wash each plate with additional 2 ml LB medium to recover    residual bacteria.-   18. Pool bacteria in a single sterile flask with a cap.-   19. Take half of the pooled bacteria and perform plasmid preparation-   20. To the remainder of the pooled bacteria, add 0.2 volumes of 80%    glycerol and mix thoroughly.-   21. Dispense 1-ml aliquots into sterile 1.5 ml microcentrifuge tubes    and freeze at −80° C.

Purify pBA-heavy chain variable domain (pBA-HC) library DNA with QIAprepSpin Miniprep kit

-   1. Resuspend pelleted bacterial cells in 500 μL P1 buffer (with    RNase A) and transfer to two microcentrifuge tubes.-   2. Add 250 μL P2 buffer and mix thoroughly by inverting the tubes    4-6 times.-   3. Add 350 μL N3 buffer and mix immediately and thoroughly by    vortexing-   4. Centrifuge for 10 min at 13,000 rpm in a microcentrifuge.-   5. Apply the supernatants to the QIAprep spin columns by decanting.-   6. Centrifuge for 1 min at 13,000 rpm in a microcentrifuge. Discard    the flow-through.-   7. Wash the QIAprep spin columns by adding 0.75 ml PE buffer.-   8. Centrifuge for 1 min at 13,000 rpm in a microcentrifuge.-   9. Discard the flow-through and centrifuge for an additional 1 min    to remove residue wash buffer.-   10. Place the QIAprep columns in a clean 1.5 ml microcentrifuge    tubes. To elute DNA, add 50 μL EB buffer to the center of each    QIAprep spin column, let stand for 1 min at room temperature.-   11. Centrifuge on 2 min at 13,000 rpm in a microcentrifuge.-   12. Determine the DNA concentration with spectrophotometer    (OD_(260/280))

BsmBI Digestion of pBA-HC Library

Prepare the following digestion reaction in a microcentrifuge tube onice:

pBA-HC library DNA (5 μg) x μL 10X NEB Buffer 3 10 μL Nuclease-freewater QS to 97 μL BsmBI (10 U/μl) 3 μL Total reaction volume 100 μL

-   1. Mix gently and spin briefly (5 sec.) in microfuge-   2. Incubate the reaction at 55° C. overnight

Day 5

-   3. Add 2 μL of Apex phosphatase to the microcentrifuge tube-   4. Incubate at 37° C. for 10 minutes-   5. Heat at 70° C. for 5 minutes to inactivate the Apex phosphatase

Purify BsmBI Digested pBA-HC Library with QIAquick PCR Purification Kit

-   1. Add 500 μL of Buffer PBI to the microcentrifuge.-   2. Mix by vortexing and quick centrifuge.-   3. Load 750 μL at a time onto a column.-   4. Centrifuge at 12,000×g for 1 minute and decant liquid from    collection tube.-   5. Repeat until all sample has been processed.-   6. Wash with 750 μL PE Buffer (Ethanol added!).-   7. Centrifuge at 12,000×g for 1 minute and decant liquid from    collection tube.-   8. Place column back onto collection tube and centrifuge again.-   9. Put column onto new microcentrifuge tubes and elute with 50 μL EB    Buffer.

Quality Control Analysis

-   1. To QC the BsmBI digested pBA-HC library, set up the following    reaction in a 1.5 ml micro-centrifuge tube:

pBA-HC-BsmBI 2 μl pBA-HC-uncut 2 μl Water 7 μl Water 7 μl Sample loadingbuffer 1 μl Sample loading buffer 1 μl Total Volume 10 μl  Total Volume10 μl 

-   2. Load 10 μl onto a 1% agarose TAE gel with 0.5 μg/ml Ethidium    Bromide. Use 1 kb plus DNA ladder as standard. Run the gel at 100V    for 20-30 minutes in 1× TAE buffer.-   3. Determine the concentration of the BsmBI digested pBA-HC library    with spectrophotometer (OD_(260/280)).-   4. Set up the following control ligation reactions to determine the    background and ligation efficiency of the BsmBI digested pBA vector:    -   c. Vector Background Ligation

pBA-HC-BsmBI (100 ng) x μL 5X T4 ligase Buffer 4 μL Nuclease-free waterQS to 19 μL T4 ligase (2,000 U/μL) 1 μL Total reaction volume 20 μL

-   -   d. Test Insert Ligations

pBA-HC-BsmBI (100 ng) x μL Test insert (5 ng, 10 ng) y μL 5X T4 ligaseBuffer 4 μL Nuclease-free water QS to 19 μL T4 ligase (2,000 U/μL) 1 μLTotal reaction volume 20 μL

-   5. Mix gently and spin briefly (5 sec.) in microfuge-   6. Incubate at room temperature for 2 hours or 16° C. overnight-   7. Transform each of the ligation reaction mixtures into XLI Blue    Supercompetent cells-   8. Pre-chill 14 ml BD Falcon polypropylene round-bottom tubes on    ice. Warm SOC medium to 42° C.-   9. Thaw the XLI Blue Supercompetent cells on ice. When thawed,    gently mix and aliquot 100 ul of cells into each of the pre-chilled    tubes.-   10. Add 1.7 ul of beta-mercaptoethanol to each aliquot of cells.    Incubate the cells on ice for 10 minutes, swirling gently every 2    minutes.-   11. Add 2 ul of the ligation reaction mixture to one aliquot of    cells. Flick the tubes gently.-   12. Incubate the tubes on ice for 30 minutes.-   13. Heat-pulse the tubes in a 42° C. water bath for 45 seconds.-   14. Incubate the tubes on ice for 2 minutes-   15. Add 900 ul of preheated SOC medium and incubate the tubes at    37° C. for 1 hour with shaking at 225-250 rpm.-   16. Plate 20 μL and 200 μL of the transformation mixture on LB agar    plates containing carbenicillin.-   17. Incubate the plates at 37° C. overnight.

Day 6

-   18. Count colonies and calculate the efficiency and background of    the vector as well as the optimal amount of insert for ligation.

Ligate Light Chain (LC) Variable Domain into BsmBI Digested pBA-HCLibrary

Prepare the following ligation reaction in a microcentrifuge tube onice:

pBA-HC-BsmBI (100 ng) x μL Light chain (LC) variable domain y μL 5X T4ligase Buffer 4 μL Nuclease-free water QS to 19 μL T4 ligase (2,000U/μL) 1 μL Total reaction volume 20 μL

-   1. Mix gently and spin briefly (5 sec.) in microfuge-   2. Incubate at room temperature for 2 hours or 16° C. overnight-   3. Transform each of the ligation reaction mixtures into XLI Blue    Supercompetent cells-   4. Pre-chill 14 ml BD Falcon polypropylene round-bottom tubes on    ice. Prepare SOC medium to 42° C.-   5. Thaw the XLI Blue Supercompetent cells on ice. When thawed,    gently mix and aliquot 100 ul of cells into each of the pre-chilled    tubes.-   6. Add 1.7 ul of beta-mercaptoethanol to each aliquot of cells.    Incubate the cells on ice for 10 minutes, swirling gently every 2    minutes.-   7. Add 2 ul of the ligation reaction mixture to one aliquot of    cells. Flick the tubes gently.-   8. Incubate the tubes on ice for 30 minutes.-   9. Heat-pulse the tubes in a 42° C. water bath for 45 seconds.-   10. Incubate the tubes on ice for 2 minutes-   11. Add 900 ul of preheated SOC medium and incubate the tubes at    37° C. for 1 hour with shaking at 225-250 rpm.-   12. Plate 20 ul and 200 ul of the transformation mixture on LB agar    plates containing Carbenicillin.-   13. Incubate the plates at 37° C. overnight.

Day 7

-   14. Count colonies on plates. Pick 48 colonies for miniprep and    sequencing.-   15. Repeat transformations to obtain at least 20,000 colonies.-   16. Add 6 ml LB medium to each plate containing colonies and gently    scrape the bacteria with a spreader for form a dense suspension.-   17. Wash each plate with additional 2 ml LB medium to recover    residual bacteria.-   18. Pool bacteria in a single sterile flask with a cap.-   19. To the remainder of the pooled bacteria, add 0.2 volumes of 80%    glycerol and mix thoroughly.-   20. Dispense 1-ml aliquots into sterile 1.5 ml microcentrifuge tubes    and freeze at −80° C.

Example 5. Transfection of Humanization Library into CHO-S Cells

This protocol describes the method of transfecting DNA into CHO-S cells.The estimated time of completion is 3 days with 384 samples per person.Table 4 shows required reagents, consumables and equipment.

TABLE 4 Reagents, Consumables, and Equipment Item Approved Catalogue #Description Supplier No. 1 Dulbecco's Modified Invitrogen 11965-092Eagle Medium 2 CD-CHO Invitrogen 10743-029 3 HT supplement Invitrogen11067-030 4 10 mM MEM Non-Essential Invitrogen 11965-092 Amino Acids 5Fetal Bovine Serum Invitrogen 26140-079 6 Lipofeectamine 2000 Invitrogen11668-027 7 PBS Invitrogen 8 Opti-MEM Reduced Invitrogen 31985-062 SerumMedium

The following buffers are required for this protocol.

-   -   Heat inactivated fetal bovine serum        -   500 ml heat inactivated fetal bovine serum in the original            vendor bottle        -   Heat for 30 minutes at 56° C. with mixing every 5 minutes        -   Prepare 50 ml aliquots and store at −20° C.    -   Serum supplemented Dulbecco's Modified Eagle Medium        -   500 ml Dulbecco's Modified Eagle Medium        -   50 ml heat inactivated fetal bovine serum        -   5 ml 10 mM MEM Non-Essential Amino Acids

Day 1

-   1. One week before transfection, transfer CHO-S cells to monolayer    culture in serum supplemented Dulbecco's Modified Eagle Medium    (D-MEM).-   2. One day before transfection, plate 0.4×10⁵ cells in 100 □l of    serum supplemented D-MEM per transfection sample in 96 well formats.

Day 2

-   3. Perform transfection at the end of the work day.-   4. For each transfection sample, prepare DNA-Lipofectamine    complexes.-   5. Dilute 0.2 μg of DNA in 25 μL Opti-MEM Reduced Serum Medium. Mix    gently.-   6. Dilute 0.5 μL Lipofectamine in 25 μL Opti-MEM Reduced Serum    Medium. Mix gently and incubate for 5 min at room temperature.-   7. Combine the diluted DNA with the diluted Lipofectamine Mix gently    and incubate for 20 min at room temperature.-   8. Add the 50 μL DNA-Lipofectamine complexes to each well containing    cells and medium. Mix gently by rocking the plate back and forth.-   9. Incubate the cells at 37° C. in a 5% CO₂ incubator overnight

Day 3

-   10. Aspirate off medium in each well. Add 100 μL of serum    supplemented D-MEM to each well. Collect supernatant for ELISA assay    and cell lysate for beta-galactosidase assay.

Example 6. Determination of Expression Levels of Humanized Clones byQuantitation ELISA

This protocol describes the method of determining the expression levelof antibodies in cell culture supernatant. The estimated time ofcompletion is 2 days with 96 samples per person. Table 5 shows requiredreagents, consumables and equipment.

TABLE 5 Reagents, Consumables, and Equipment Item Approved Catalogue #Description Supplier No. 1 Tween-20 Invitrogen 11965-092 2 Carnationnon-fat milk Local supermarket 3 PBS Irvine 9242 Scientific 4 anti-humanIgG(H + L)-HRP Promega W4031 5 Human IgG Invitrogen 12000C 6 Nunc-ImmunoMaxisorp 96 Nalge Nunc 439454 well plates 7 Affinity-purified Fc- Sigma12136-1 ml specific goat anti-human IgG 8 3,3′,5,5′- Sigma T4444Tetramethylbenzidine Liquid Substrate

The following buffers are required for this protocol.

-   -   Washing solution        -   0.05% Tween−20 in PBS    -   Blocking solution        -   2% Carnation non-fat milk in PBS

Day 1

-   11. Coat Nunc-Immuno Maxisorp 96 well plates with 100 μL of 10 μg/ml    affinity-purified Fc-specific goat anti-human IgG in coating    solution.-   12. Cover plates with sealers and incubate overnight at 4° C.

Day 2

-   13. Decant plates and tap out residue liquid.-   14. Add 200 ul washing solution. Shake at 200 rpm for 5 min at room    temperature.-   15. Decant plates and tap out residue liquid.-   16. Add 200 ul blocking solution. Shake at 200 rpm for 1 hour at    room temperature.-   17. Decant plates and tap out residue liquid.-   18. Add duplicates of 100 ul/well of standardized concentration of    purified human serum IgG in blocking solution to the plates.-   19. Add duplicates of 100 ul of supernatant from the transfection    procedure to the plates.-   20. Shake at 200 rpm for one hour at room temperature.-   21. Decant plates and tap out residual liquid.-   22. Add 200 ul washing solution. Shake at 200 rpm for 5 min at room    temperature.-   23. Repeat step 11-12 3 times.-   24. Add 100 ul of 1:5000 dilution of affinity purified goat    anti-human antibody conjugate with HRP in blocking solution to each    well.-   25. Shake at 200 rpm for one hour at room temperature.-   26. Decant plates and tap out residual liquid.-   27. Add 200 ul washing solution. Shake at 200 rpm for 5 min at room    temperature.-   28. Repeat step 17-18 3 times.-   29. Add 100 ul of Sigma TMB substrate to each well. Incubate at room    temperature and check every 2-5 minutes.-   30. Add 100 ul 1N HCl to stop the reaction.-   31. Read at 450 nm.

Example 7. Determination of Affinity of Humanized Clones by AffinityELISA

This protocol describes the method of comparing the affinity ofantibodies in cell culture supernatant. The estimated time of completionis 2 days with 96 samples per person. Table 6 shows required reagents,consumables and equipment.

TABLE 6 Reagents, Consumables, and Equipment Item Approved Catalogue #Description Supplier No. 1 Tween-20 Invitrogen 11965-092 2 Carnationnon-fat milk Local supermarket 3 PBS Irvine 9242 Scientific 4 anti-humanIgG(H + L)-HRP Promega W4031 5 Control antibody Project dependent 6Nunc-Immuno Maxisorp Nalge Nunc 439454 96 well plates 7 Antigen Projectdependent 8 3,3′,5,5′- Sigma T4444 Tetramethylbenzidine Liquid Substrate

The required buffers for this protocol are as described in Example 6.

Day 1

-   32. Coat Nunc-Immuno Maxisorp 96 well plates with 100 μL of 2 μg/ml    antigen in coating solution.-   33. Cover plates with sealers and incubate overnight at 4° C.

Day 2

-   34. Decant plates and tap out residue liquid.-   35. Add 200 ul washing solution. Shake at 200 rpm for 5 min at room    temperature.-   36. Decant plates and tap out residue liquid.-   37. Add 200 ul blocking solution. Shake at 200 rpm for 1 hour at    room temperature.-   38. Decant plates and tap out residue liquid.-   39. Add duplicates of 100 ul/well of control antibody (2 μg/ml) in    blocking solution to the plates.-   40. Add duplicates of 100 ul of supernatant from transfection (SOP    5A) to the plates.-   41. Shake at 200 rpm for one hour at room temperature.-   42. Decant plates and tap out residual liquid.-   43. Add 200 ul washing solution. Shake at 200 rpm for 5 min at room    temperature.-   44. Repeat step 11-12 3 times.-   45. Add 100 ul of 1:5000 dilution of affinity purified goat    anti-human antibody conjugate with HRP in blocking solution to each    well.-   46. Shake at 200 rpm for one hour at room temperature.-   47. Decant plates and tap out residual liquid.-   48. Add 200 ul washing solution. Shake at 200 rpm for 5 min at room    temperature.-   49. Repeat step 17-18 3 times.-   50. Add 100 ul of Sigma TMB substrate to each well. Incubate at room    temperature and check every 2-5 minutes.-   51. Add 100 ul 1N HCl to stop the reaction.-   52. Read at 450 nm.

Example 8. Biacore (Surface Plasmon Resonance) Affinity Measurement ofBA001 and Humanized Derivatives

BIAcore 3000, GE Healthcare, was used to determine binding curves andkinetic parameters. An anti-human Fc (1.8 mg/ml) was diluted to aconcentration of 50 ug/ml in NaOAc buffer (10 mM, pH 4.8) and coupled tothe carboxymethylated dextran matrix of a CM-5 sensor chip using themanufacturer's amine-coupling chemistry as described in the BIAcoresystems manual. Using the surface preparation wizard aiming for 10000RU, the carboxyl groups on the sensor surfaces were first activated withNHS/EDC followed by the addition of the anti-human Fc. The remainingactivated groups were blocked by the injection of 1M ethanolamine Eachof the flow cells was coupled individually. Employing these conditions,the four flow cell surfaces containing 7554-9571 RU of anti-human Fcwere prepared. In preliminary experiments, it was determine that threeinjections (15 ul at 30 ul/min) 100 mM H₃PO₄/0.05% CHAPS wouldefficiently remove the bound immunoglobulin and preserve the bindingcapacity of the immobilized anti-human Fc.

Experiments were performed on the BIAcore 3000 at 25° C. and a flow rateof 30 ul/min. The antibody candidate was dissolved in HBS (10 mM HEPESwith 0.15M NaCl, 3.4 mM EDTA, and 0.05% surfactant P20 at pH 7.4) at 5ug/ml. The analyte, IL-6, was dissolved in HBS at 0.25, 0.125, 0.062,0.031 and 0.015 ug/ml. 3*30 ul of 5 ug/ml of antibody BA001 was flowedover its respective flow cell followed by injections of 240 ul of eachIL-6 concentration at 30 ul/min (association phase) and an uninterrupted1200 seconds of buffer flow (dissociation phase). The surface of thechip was regenerated by three sequential injections of 15 ul each with100 m M H₃PO₄/0.05% CHAPS. The injections of HBS serve as a reference(blank sensogram) for the subtraction of bulk refractive indices foranalysis. Using the 1:1 model in BIAevaluation 4.1, both a local fit andglobal fit was done for both dissociation (kd, [s−1] and association(ka, [M⁻¹s⁻¹]) and the dissociation constant (KD, [M]) calculated(kd/ka).

Analysis was done using BIAeveluation version 3.0. Kinetic constantswere derived from sensogram data by fitting the experimental curves tothe rate equations derived from models of the interaction mechanisms. Aglobal analysis using a 1:1 binding model with local RUmax fit, the ka,kd, and KD were determined.

The following equations were utilized:

$\left. {{Ab} + {Ag}}\rightleftharpoons{AbAg} \right.{K_{a} = {\frac{\lbrack{AbAg}\rbrack}{\lbrack{Ab}\rbrack\lbrack{Ag}\rbrack}{\frac{1}{K_{d}}.}}}$

Biacore data for humanized anti-IL6 Mabs before affinity maturation isset forth in FIG. 4.

Example 9. Humanized Antibody Preparation from a Template Anti-IL6Antibody

Sequences for the template antibody and lead hits and fragment nucleicacid and protein sequences are disclosed in US Publication No.2010/0138945, Humanized Anti-IL-6 Antibodies, Frey et al., which isincorporated herein by reference in its entirety. In this example, thetemplate antibody was BA001; an anti-human IL-6 antibody. Templateantibody BA001 is the same sequence, but manufactured in a differentexpression system, compared to CNT0328, a chimeric, human-murineantibody from US 2006/0257407, which is incorporated herein byreference.

FIG. 1 shows a schematic of the method analogous to that used forhumanization of the template antibody BA001. Detailed steps aredescribed in Examples 1-8. The template antibody was cloned, andsequences were identified for each complementarity determining region(CDR). The original CDRs were synthesized; and a synthetic ds DNAfragment library based upon each CDR was prepared. A human frameworkpool was prepared only from human functionally expressed antibodiesobtained from human germline sequences. The CDRs and the humanframeworks were assembled, in this case by step wise liquid phaseligation, to form a humanized light chain (LC) variable domain encodinglibrary; and a humanized heavy chain (HC) variable domain encodinglibrary. LC and HC domains were inserted to a vector. In one aspect, thevector contains the sequence which encodes LC and HC framework 4. Thevector with the insert is transfected and expressed in a mammalian cellline to prepare a full length human antibody variant library; in thiscase a human IgG variant library. The library was screened to selecthumanized antibodies which were comparable or superior to the donorantibody in at least one characteristic, for example antigen affinity orexpression level in a mammalian production cell line.

FIG. 2 shows data from the primary screen of humanized variants oftemplate antibody BA001. The primary screen comprised high throughputELISA of variants compared to the donor antibody, shown with anasterisk. ELISAs were used to determine antigen binding andquantitation. ELISA protocols are presented in detail in the Examples.

FIG. 3 shows the top 8 confirmed humanized antibody variant hits interms of both expression and function compared to the template antibodyBA001. Selection of hits was based on antigen binding, expression levelin a mammalian cell line and sequence diversity. DNA and proteinsequences for BA001 and humanized variants shown are described in U.S.Publication No. US 2010/0138945, which is incorporated herein byreference.

FIG. 4 shows binding affinity BiaCore surface plasmon resonance data forhumanized anti-IL6 antibodies compared to a template antibody. Data forthe template, CNT0328, a chimeric, human-murine antibody, is from US2006/0257407, which is incorporated herein by reference. BA001 is also atemplate antibody that has the same sequence as the template CNT0328,but was manufactured in a different expression system. Humanized variantantibodies h1-h8 were obtained with no additional affinity maturation.

FIG. 5 shows results of an ELISA wherein humanized antibody variantsblock antigen binding of template antibody BA001.

It will be clear that the invention can be practiced otherwise than asparticularly described in the foregoing description and examples.Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims

We claim:
 1. A method of producing a humanized antibody or a humanizedantibody fragment, the method comprising: (a) synthesizingimmunoglobulin heavy chain double stranded DNA fragment librariescomprising complementarity determining region fragment encodinglibraries and framework fragment encoding libraries, wherein at leastone complementarity determining region fragment library includes atleast one double stranded DNA fragment encoding at least a portion of aheavy chain complementarity determining region having at least 90%sequence identity to a complementarity determining region of a non-humantemplate antibody and each framework fragment library includes at leastone double stranded DNA fragment encoding at least a portion of a heavychain framework derived from a human framework pool obtained fromfunctionally expressed human antibodies having framework regions thathave a sequence identity of at least 85% with a framework region of thetemplate antibody; (b) synthesizing immunoglobulin light chain doublestranded DNA fragment libraries comprising complementarity determiningregion fragment encoding libraries and framework fragment encodinglibraries, wherein at least one complementarity determining regionfragment library includes at least one double strand DNAed fragmentencoding at least a portion of a light chain complementarity determiningregion having at least 90% sequence identity to a complementaritydetermining region of the template antibody and each framework fragmentlibrary comprising at least one double stranded DNA fragment encoding atleast a portion of a light chain framework derived from a humanframework pool obtained from functionally expressed human antibodieshaving framework regions that have a sequence identity at least 85% witha framework region of the template antibody; (c) assembling from theheavy chain fragment libraries by stepwise liquid phase ligation ofheavy chain framework encoding fragments from the heavy chain frameworkfragment encoding libraries and heavy chain complementarity determiningregion encoding fragments from the heavy chain complementaritydetermining region fragment encoding libraries in the order of:framework1-complementarity determiningregion1-framework2-complementarity determiningregion2-framework3-complementarity determining region3 to produce ahumanized heavy chain variable domain encoding library; (d) assemblingfrom the light chain fragment libraries by stepwise liquid phaseligation of light chain framework encoding fragments from the lightchain framework fragment encoding libraries and light chaincomplementarity determining region encoding fragments from the lightchain complementarity determining region fragment encoding libraries inthe order of: framework1-complementarity determiningregion1-framework2-complementarity determiningregion2-framework3-complementarity determining region3 to produce ahumanized light chain variable domain encoding library; (e) cloning theassembled humanized heavy chain variable domain encoding library and theassembled light chain variable domain encoding library into anexpression vector to create a humanization library; (f) transfecting thehumanization library into cells; (g) expressing humanized antibodies orhumanized antibody fragments in the cells to create a humanized antibodylibrary; and (h) screening the humanized antibody library to determinean affinity of the humanized antibodies or humanized antibody fragmentsfor an antigen compared to an affinity of the template antibody to thesame antigen.
 2. The method of claim 1 wherein the expression vectorcomprises a nucleic acid sequence encoding heavy chain framework region4.
 3. The method of claim 2 wherein the nucleotide sequence encodingheavy chain framework 4 is derived from a human heavy chain variabledomain of a functionally expressed human antibody.
 4. The method ofclaim 1 wherein the expression vector comprises a nucleic acid sequenceencoding light chain framework region
 4. 5. The method of claim 4wherein the nucleotide sequence encoding light chain framework 4 isderived from a human light chain variable domain of a functionallyexpressed human antibody.
 6. The method of claim 1 wherein the humanizedantibody library has 10,000,000 members or fewer.
 7. The method of claim6 wherein the humanized antibody library has 1,000,000 members or fewer.8. The method of claim 7 wherein the humanized antibody library has100,000 members or fewer.
 9. The method of claim 1 wherein the cloningstep comprises cloning the assembled humanized heavy chain variabledomain encoding library into the expression vector to create avector-heavy chain variable domain DNA library, and ligating theassembled light chain variable domain encoding library into thevector-heavy chain carriable domain DNA library to create thehumanization library.
 10. The method of claim 1 wherein the expressionstep comprises expressing both the humanized heavy chain variable domainand the humanized light chain variable domain from a single promoter.11. The method of claim 1 further comprising a step of screening for ahumanized antibody or humanized antibody fragment having one or moreadditional improved characteristics when compared to the templateantibody; the one or more characteristics selected from the groupconsisting of: equilibrium dissociation constant K_(D); stability;melting temperature T_(m); pI; solubility; expression level; reducedimmunogenicity and improved effector function relative to the templateantibody.
 12. The method of claim 1 wherein the cells are selected froma eukaryotic cell production host cell line selected from 3T3 mousefibroblast cells; BHK21 Syrian hamster fibroblast cells; MDCK, dogepithelial cells; Hela human epithelial cells; PtK1 rat kangarooepithelial cells; SP2/0 mouse plasma cells; and NS0 mouse plasma cells;COS monkey kidney cells; CHO, CHO-S Chinese hamster ovary cells; R1mouse embryonic cells; E14.1 mouse embryonic cells; PER C.6, humanembryonic cells; S. cerevisiae yeast cells; and picchia yeast cells. 13.The method of claim 12 wherein the eukaryotic cell production host cellline is CHO-S.
 14. The method of claim 12 wherein the eukaryotic cellproduction host cell line is CHOK1SV or NS0.
 15. The method of claim 1wherein the cell is a eukaryotic cell production host with antibody cellsurface display.
 16. The method of claim 15 wherein one or both of thescreening steps is performed in the eukaryotic cell production host. 17.The method of claim 1 wherein one or both of the screening steps areselected from quantitative ELISA; affinity ELISA; ELISPOT; flowcytometry, immunocytology, Biacore® surface plasmon resonance analysis,Sapidyne KinExA™ kinetic exclusion assay; SDS-PAGE; Western blot, andHPLC.
 18. The method of claim 1, wherein the at least one double strandDNA fragment encoding at least a portion of a heavy chaincomplementarity determining region is derived from the template antibodythrough evolving a complementarity determining region of the templateantibody.
 19. The method of claim 1, wherein the at least one doublestrand DNA fragment encoding at least a portion of a light chaincomplementarity determining region is derived from the template antibodythrough evolving a complementarity determining region of the templateantibody.
 20. The method of claim 18, wherein the evolving acomplementarity determining region is accomplished by substitutions,insertions and deletions.
 21. The method of claim 18, wherein theevolving a complementarity determining region is accomplished byComprehensive Positional Evolution (CPE™), Comprehensive ProteinSynthesis (CPS™), Flex Evolution, Synergy Evolution, ComprehensivePositional Insertion evolution (CPI™), or Comprehensive PositionalDeletion evolution (CPD™).
 22. The method of claim 1, wherein the atleast a portion of a heavy chain complementarity determining region hasa sequence at least 90% identical to a complementarity determiningregion of the template antibody.